Table of Contents
Acknowledgments
9
Chapter 1. Introduction: post-operative adhesion formation
11
A. Clinical significance of post-operative adhesions
12
B. Pathophysiology of adhesion formation
13
I. The classic model: a local phenomenon between opposing lesions
13
II. The updated model: the important role of the peritoneal cavity
14
C. Prevention of adhesion formation
18
D. Important clinical factors related to the pneumoperitoneum
22
I. CO2 absorption during laparoscopy
22
II. Nitrous oxide pneumoperitoneum in laparoscopy
23
III. Tumor implantation in laparoscopy
24
IV. Post-operative pain and inflammation in laparoscopy
25
E. Aims of the thesis
Chapter 2. General materials and methods
26
29
A. The laparoscopic mouse model
29
B. Clinical trials
32
I. Adding 4% oxygen to the pneumoperitoneum
33
II. Full conditioning of the peritoneal cavity
33
C. Statistics
36
Chapter 3. Results
37
A. Animal experiments
37
I. Establishing a learning curve
37
II. Mesothelial cells in prevention of adhesions
40
III. Tumor implantation
44
IV. Nitrous oxide
49
V. Depth of hypoxia
51
B. Clinical trials
I. Adding 3% oxygen to the pneumoperitoneum
53
53
a.CO2 absorption
53
b. Pain and inflammation
56
5
c. Port site metastases
60
d. Ultramicroscopic alterations
61
e. Simultaneous analysis of inflammation and surgery
related complications when 4% of oxygen is added
to the CO2pneumoperitoneum
II. Full conditioning of the peritoneal cavity
63
65
a. CO2 absorption
65
b. Pain and inflammation
66
c. Adhesion prevention
69
d. Absorption of fluid from the peritoneal cavity
72
III. Lavage of the peritoneal cavity
77
IV. pH of irrigation fluid and wet tongue-tip pain
79
Chapter 4. Discussion and conclusions
A. Understanding the peritoneal cavity
81
81
I. The mesothelial layer: a fragile border
81
II. Mesothelial cells in prevention of adhesions
83
III. Nitrous oxide
85
IV. Training and experimental models
86
B. Translation to clinical practice
87
I. Addition of oxygen
87
II. Full conditioning of the peritoneal cavity
88
III. Oncologic implications
89
C. Conclusions and future perspectives
93
D. Summary - English
95
E. Summary - Dutch
97
Chapter 5. References
99
Chapter 6. Bibliography, and curriculum vitae of Jasper Verguts
111
Chapter 7. Addenda
117
6
If your experiment needs statistics, then you ought to have done a better experiment.
Ernest Rutherford (1871- 1937)
To my sister and inspiration for life, Eva
7
8
Acknowledgements
This thesis is respectfully presented to the Katholieke Universiteit Leuven through its rector
prof. dr. M. Waer and its vice-rector and chairman of Biomedical Science prof. dr. M.
Casteels, to the Faculty of Medicine through its dean prof. dr. B. Himpens, and to the
Department Women and Child through its chairman prof. dr. J. Deprest.
I am grateful to the members of the jury, prof. dr. F. Penninckx, prof. dr. P. Moerman, prof.
dr. H. Van Goor (Nijmegen) and prof. dr. A. Wattiez (Strasbourg,) for their most valuable
remarks and critical reading of the manuscript and to the chairman of the jury prof. dr. F.
Amant. Thanks to prof. dr. D. Deridder who chaired the reading committee and for guiding
me through the final mazes of the doctoral net.
I want to thank my promoter, prof. dr. Philippe R. Koninckx, the engine that kept me going
throughout my doctoral thesis and thought me there is more in life than gynecology. He
guided me on every investigational road I took and made me take control off my own goals.
His enthusiasm, perseverance and most of all strive for quality made me a better scientist.
I would like to thank my co-promoter prof. dr. Ignace Vergote for supporting me in my first
steps in gynecology now some twelve years ago. He improved my surgical skills in
gynecology which will be mine for the rest of my career.
I am grateful to all my colleagues, prof. dr. Myriam Hanssens, prof. dr. Jan Deprest, prof dr.
Dirk Timmerman, prof. dr. Willy Poppe and dr. Anne Pexsters who relieved me of some of
my clinical and other duties to be able to work on my thesis or gave me advise which I could
use all the same. Thanks to Marleen Craessaerts and Diane Wolput for being there when I
needed you to answer practical and unpractical questions. They are (probably) the best trial
team in the world.
I would also like to thank my sister Mieke Verguts for helping me preparing this manuscript.
Great job!
9
A special thanks to prof. dr. Bernard Vanacker, Roberta Corona, An Coosemans, Mercedes
Binda, Carlo De Cicco, Karina Mailova, and Tom Bourne for substantial help in our trials and
sharing thoughts on design and results which cleared the way for even more trials. A
perpetuum mobile which I am grateful to be part of. Many thanks to all working in the lab
for guiding me and making the experiments possible. You’ve opened a new world to me.
Thanks to Sandra Meeus and her team from the A-cluster in the operating theater for
making the impossible possible, Chris Verheyden and Mark Declerck from the technical
department, Eugeen Steurs from Storz®, and Thomas Koninckx and Bob Koninckx from
eSaturnus®.
Thanks to my dearest friends Wim Vandoninck and Gert Vangeel for supporting me through
some difficult years. Thanks to all I ever met, this thesis is the result of a number of events
you where definitely part of.
Where it all comes down to are you, my family and my loving and caring wife Sandra and our
four most precious gifts to this world: Lisa, Anne, Marit and Andreas. All this means nothing
compared to what you mean to me.
Leuven, June 2011
10
Chapter 1
Introduction: post-operative adhesion formation
Adhesions are fibrous bands between tissues and organs and are one of the most
underestimated problems which may occur following surgery. The term “adhesions” comes
from the Latin “addere” which means “to add something”. Adhesions are therefore not
restricted to one type of organ or tissue, but can involve any kind of tissue or even foreign
material.
There are two different classifications of adhesions that are commonly used. First there is
the classification by Pouly et al.1 which recognizes three types of adhesions. Adhesions
occurring between injured areas are classified as “adhesions”. “De novo adhesions” occur at
an area outside the surgical trauma site where no previous adhesions occurred. Adhesions at
a site where previous adhesions were lysed are classified as “adhesion reformation”. The
classification of Diamond et al. recognizes two types of adhesions: “de novo adhesions” and
“adhesion reformation” as Pouly et al., but sub classifies them depending on whether
adhesions occur at or outside the surgical site2.
Until recently adhesions were documented to be an almost unavoidable consequence of
abdominal and pelvic surgery, as they occurred in as much as 93% of all patients undergoing
abdominal surgery3. It was shown during autopsy that in up to 28% of patients who never
underwent surgery also developed adhesions, as they may be caused for example by pelvic
inflammatory disease or endometriosis4.
As adhesions may cause pain, infertility and/or bowel obstruction, treatment can be
necessary. Treatment of adhesions may fail since adhesions can be difficult to remove or,
when removed, tend to reform. Therefore prevention (primary and secondary) is of utmost
importance. Primary prevention consists in preventing tissue damage. Secondary prevention
of adhesions consists in treating already inflicted tissue damage. Gentle tissue handling is
thought to be a form of primary prevention. The use of barriers to seal off the damaged area
is thought to be a form of secondary prevention. The focus on primary or secondary
prevention only gained interest with the understanding of the mechanisms that cause
adhesions, and with the ability to prevent adhesions efficiently with some basic surgical
principles (e.g. gentle tissue handling, avoiding desiccation). These principles were only later
11
evaluated in animal and clinical trials, but served well for the time being. Laparoscopy, when
introduced in the eighties, was also thought to be beneficial in the prevention of adhesions
as it is considered minimal invasive. However, the positive effect of this new approach on
adhesion prevention was not as good as expected.
A. Clinical significance of post-operative adhesions
A study published in Digestive Surgery showed that adhesions developed in more than 90%
of patients who underwent open abdominal surgery and in 55%–100% of women who
underwent pelvic surgery5. Adhesions from prior abdominal or pelvic surgery can decrease
visibility and access at subsequent abdominal or pelvic surgery. In a very large study (29,790
participants) published in The Lancet, 35% of patients who underwent open abdominal or
pelvic surgery were readmitted to the hospital on an average of two times after their surgery
due to adhesion-related or adhesion-suspected complications6. Over 22% of all readmissions
occurred in the first year after the initial surgery and was linear over time. In the SCAR trial it
was demonstrated that the risk of readmission due to adhesions was 5% over a ten year
period following an initial open surgical procedure for a gynecological condition7. Of the
readmissions about 40% was readmitted between two and five times. This suggests that a
great number of adhesions formed after surgery occur without symptoms.
In 2001 Parker et al. published a study based on the same population group as Lower et al.,
but they concentrated their analysis on those patients who had lower gastro-intestinal
surgery8. In this cohort (32.6%) patients were readmitted on an average of 2.2 times in the
subsequent 10 years for a potential adhesion-related problem (twice as high as in the first
study). Although 25.4% of readmissions occurred in the first postoperative year, they
steadily increased throughout the study period. After open lower abdominal surgery 7.3% of
readmissions were directly related to adhesions. The overall average rate of readmissions
was 70.4 per 100 initial operations, with 5.1 directly related to adhesions.
Major complications of adhesions will depend on localization of the adhesion, causing
chronic pelvic pain (cfr. infra), bowel obstruction or infertility9;10. Adhesion-related
complexity at re-operation added significant risk to subsequent surgical procedures11.
12
The high clinical significance of adhesions however will not always prompt practitioners to
discuss the matter with their patients.
B. Pathophysiology of adhesion formation
I. The classic model: a local phenomenon between opposing lesions
Adhesion formation is mediated through different mechanisms. (Figure 1) Damage to
peritoneal surfaces induces a response starting with an acute inflammatory reaction, and a
process involving mesothelial cells, macrophages, exudate with cytokines and coagulation
factors, neutrophils and leukocytes12. The inflammatory response directly results in fibrin
deposition at the lesion within the first hours after the peritoneal trauma with a peak on
postoperative day four to five. Mesothelial cells obviously play an important role in
peritoneal repair. Within hours a peritoneal defect (i.e. caused by a trauma during surgery) is
covered with macrophages and mesothelial cells, previously described as tissue repair
cells13.
Figure 1: the classic model
13
The healing process starts from multiple islands over the defect from where mesothelial cells
proliferate. Hence small and large defects heal in the same short time from seven to ten
days14. Mesothelial cells at the edge of a lesion, and mesothelial cells of the opposing
mesothelial surface develop an increased dividing capacity, a phenomenon being maximal
within two days, which suggest locally secreted factors15. If mesothelial cells are capable in
covering the lesion, then fibrinolysis will complete within a few days with resorption of
degradation products and reepithelialization will result in a smooth healed tissue surface.
If the normal rapid repair of peritoneal lesions fails or when repair is delayed, other
processes which were activated may become dominant. Within 4 to 6 days fibroblasts
invading the fibrin scaffold start to proliferate, and angiogenesis starts, leading invariably to
adhesion formation. The importance of the fibrin scaffold between 2 injured surfaces was
elegantly demonstrated since by separating these areas by silastic membranes for as little as
30 hours abolished adhesion formation16. This type of experiments reinforced the belief that
adhesion formation is a local process and that prevention should aim at separating the
surfaces for at least 2 days. Medical treatment given intravenously or intraperitoneally has
thus been considered less important since this type of treatment would have difficulties to
reach the injured zone because of local ischemia after coagulation and since it is shielded by
the fibrin plug. The pathophysiology of this local process has been considered an
inflammatory reaction, with players and mechanisms as fibrinolysis, plasmin activation and
PAI’s, local macrophages and their secretion products and the overall oxygenation of the
area or the absence thereof, driving angiogenesis, fibroblast proliferation and mesothelial
repair. This concept of a local process is widely supported by reduced adhesions with
administration of antibodies against PAI-116 or fibrinolytic agents17, neutralizing antiserum
against VEGF, antibodies against PlGF and against VEGFR-118.
II. The updated model: the important role of the peritoneal cavity
Traditionally the peritoneum is considered a tissue, reducing friction between organs. It’s
the largest ‘organ’ of the body lining the peritoneal cavity with visceral and parietal surfaces
covering the internal organs and body wall, respectively. It is considered as the epithelium of
mesodermal cavities and it has mesenchymal and epithelial properties.
14
Two types of cells cover the peritoneal surface: squamous cells and cuboïdal cells. Squamous
cells are the predominant type and have some degree of active fluid and molecule transport.
Cuboïdal cells are predominant at areas of injury, at milky spots of the omentum and at the
peritoneal side of the diaphragm overlaying the lymphatic lacunae. They are specialized in
leucocyte transport, synthesis of cytokines, growth factor and CA-125 and control of
coagulation and fibrinolysis by secretion of plasminogen activator inhibitor (PAI)-1 and -2
and plasminogen activators (PA) urokinase PA (uPA) and tissue PA (tPA).
Transport of proteins over this mesothelial membrane is low for larger proteins, explaining
low concentrations of albumin and fibrinogen in the peritoneal fluid and low plasma
concentrations of CA 12519;20.
Following serosal injury and the subsequent inflammation with production of exudate, there
is a fine balance between the secreted proteins, such as fibrinogen and plasminogen, which,
if disrupted may result in the formation of adhesions. This exudate has similar
concentrations of proteins as the plasma.
The origin of the mesothelial cells involved in the repair of a serosal injury (cfr. supra)
remains somehow unclear. Possible origins are macrophages which could transform into
mesothelial cells when in contact with a defect, subserosal mesothelial cells which could
migrate into the wound or free-floating mesothelial which cells could implant. These new
mesothelial cells could eventually originate from bone marrow precursors21. Indeed,
subserosal mesothelial cells are pluripotent, since they can differentiate to mesenchymal
and epithelial cells. After peritoneal injury they differentiate and start expressing cytokeratin
while losing expression of vimentin22;23. The exudate after injury contains a high number of
fibroblast and epitheloid like cells. These cells have low proliferative activity for at least 48
hours24. That free floating mesothelial cells are important, is attractive since they are
present at all times and since their number increases twelve times within in two to five days
after injury25, since these cells were demonstrated to implant at and since extensive lavage
with removal of these free floating cells slows down peritoneal healing26. That mesothelial
cells might originate from bone marrow precursors in blood is probably less important since
total body radiation has no effect upon peritoneal wound healing27.
Intraperitoneally injected exogenous mesothelial cells can implant into the injured area and
reduce adhesion formation in the human by injection of cultured mesothelial cells obtained
from omentum28. This confirms the observations in a staphylococcal peritonitis rabbit
15
model29;30. In a rat abrasion model the injection of cultured mesothelial cells or
mesenchymal stem cells derived from skeletal muscles of newborn rats decreased adhesion
formation28;31. Recently intraperitoneal implantation of an artificial peritoneum with
collagen gel, fibroblasts and overlying mesothelial cells on an injured site significantly
reduced adhesions32. Mesothelial cells grown from adhesions moreover are different form
fibroblast derived from normal peritoneum33.
The entire peritoneal cavity is exposed to the laparoscopic gas and to air during laparotomy
and the mesothelial cells are thus influenced as homeostasis is disrupted. The peritoneal pH
seemed to immediately decrease during insufflation of CO2 into the peritoneal cavity, a
response that might influence biological events. This peritoneal effect also seemed to
influence systemic acid-base balance, probably due to trans-peritoneal absorption34;35.This
acidification of the peritoneal cavity whether by abdominal insufflation or by experimental
peritoneal acid lavage increased serum IL-10 and decreased serum TNFalpha levels in
response to a systemic lipopolysaccharide challenge mimicking a septic status. The degree of
peritoneal acidification correlated with the degree of inflammatory response reduction and
these results supported the hypothesis that pneumoperitoneum-mediated attenuation of
the inflammatory response after laparoscopic surgery occurs via a mechanism of peritoneal
cell acidification36. The direct relation between CO2 insufflation, acidification of the
peritoneum and a decreased immunoprotection might thus result in an altered adhesion
formation. The reduction of a systemic immune response was already shown by Vittimberga
et al. who conducted a systemic review on CRP and IL-6 after laparotomy and laparoscopy37.
During the last decade the entire peritoneal cavity has been shown to be a cofactor in
adhesion formation thus intervening with the local phenomena of peritoneal repair.
Identified so far are hypoxia of the mesothelial cells due to CO2 pneumoperitoneum,
desiccation of cells or tissue manipulation38-40. CO2 Pneumoperitoneum has been
demonstrated to increase adhesions and this increase is time- and pressure-dependent41. It
is thought that these factors damage mesothelial cells altering mesothelial cell
morphology42;43 and general structure of the mesothelial layer: trauma to the large and flat
mesothelial cells will induce them to retract as a defense mechanism leading to mechanisms
through which adhesion formation is further modulated. We can only speculate that
macrophages and their secretion products, blood constituents or other inflammatory
products affect directly the repair process or the differentiation of mesenchymal cells at the
16
injured area. Peritoneal hypoxia was suggested as a driving mechanism41;44;45 since
pneumoperitoneum-enhanced adhesions could be reduced by addition of 2-4% of O2 to the
CO2 pneumoperitoneum in various animal models (rabbits, mice). It was shown that the
mesothelial layer then stains hypoxic and since the increase in adhesions is prevented by the
addition of 3-4% of oxygen (restoring the physiologic intraperitoneal partial oxygen pressure
of 30 to 40 mm Hg), and is absent in mice deficient for HIF1a or HIF2a, hypoxemia response
factor being the first to be activated by hypoxia. This concept was supported by the
observed decrease in adhesions in mice deficient for the hypoxia-inducable factors (HIF)
1alfa and 2alfa, VEGF A and B and PlGF40;46. Hypothermia also decreases adhesion
formation43;47 and this was demonstrated for cooling and cooling associated with
desiccation39.
Another aspect of adhesion formation during laparoscopy is an increase in Reactive Oxygen
Species (ROS) as a result of an ischemia-reperfusion process during insufflation and
deflation48;49. Adhesions can be reduced by the use of ROS scavengers50. Induction of ROS by
laparoscopy and the subsequent acidosis is comparable to the ischemic preconditioning
which has been studied over the last two decades in coronary occlusion models where
ischemia and reperfusion may activate a cascade of events leading to the death of
myocardial cells. There is agreement that reactive oxygen species production by the
mitochondria is an essential part in the protective mechanism of ischemic preconditioning51.
Even brief ischemic preconditioning puts the cardiac cells in a protective phenotype for
several hours, but this mechanism is not well understood today. This mechanism of
protection is only exerted in the reperfusion phase, not in the ischemic phase. We noted
that ROS scavengers protected against adhesion formation in a laparoscopic mouse model
exposed to a carbon dioxide pneumoperitoneum52. I believe that the ischemic condition of a
carbon dioxide pneumoperitoneum may stimulate ROS production and only demonstrate a
protective effect in the reperfusion phase as was demonstrated by Kece et al. who noted
that adhesions reduced when laparotomy was preceded by laparoscopy 53. A continuous
ischemic phase will not display any protective effects from the ROS formed.
17
C. Prevention of adhesion formation
Adhesion prevention would suggest a comprehension of adhesion formation, but
recognition of the principles of good surgical practice started with the introduction of
microsurgery formation without proper studies supporting gentle tissue handling,
comprising moistening of tissues by continuous irrigation, moistening of abdominal packs,
glass or plastic rods for mobilization of tissues, and precise micro-instruments. it is only
recently that the importance of prevention of desiccation and of gentle tissue handling have
been proven, emphasizing how important and accurate clinical observation can be 38.
Comprehension of adhesion formation by formal animal experiments the former two
decades made introduction of further steps in the prevention of adhesions possible.
Extrapolation of data from animal research to the human implies however the believe that
the detrimental effect of a CO2 pneumoperitoneum, the duration dependent increased CO2
resorbtion as observed in mice and in rabbits, also occurs in human.
Considering primary prevention of adhesions with prevention of peritoneal damage, animal
models suggest reduction of mesothelial hypoxia and dessication by addition of 3-4% of
oxygen en maximal humidification of the insufflation gas39. Moreover, cooling of the
peritoneal cavity is important since cells become more resistant to metabolic damage at
lower temperatures54. Other important considerations are a reduction in operation time,
reduction in blood loss and the extent of tissue manipulation.
Generally when foreign material comes into contact with the peritoneum an inflammatory
reaction takes place provoking adhesion formation. This is observed in cases of infections
due to trocar insertion through an infected umbilicus of when opening the vagina during
laparoscopic surgery. This is even more likely with entry into the bowel. Whether extensive
lavage following surgery might reduce adhesion formation or the risk of some minor
infection is unknown. Following deep endometriosis surgery with full thickness resection and
a bowel suture, extensive lavage with 8 liters clearly decreased the postoperative
inflammation as judged by CRP concentrations while preventing late bowel perforations (De
Cicco et al. submitted)
18
Taken together these principles of good surgical practice with conditioning and cooling of
the pneumoperitoneum and prevention of inflammation a reduction in adhesion formation
by 60% could be obtained.
Secondary prevention of adhesion formation focuses on restoring already damaged
mesothelial tissue. This can be achieved by using gels, sheets or by instilling fluids keeping
the organs separated until complete healing of the peritoneum or by medication altering the
inflammation pathway leading to adhesion formation. A wide range of products has been
shown to be effective in animal models. Efficacy of all products described so far has been
extensively investigated in our laparoscopic mouse model. It should be realized that in this
model all criteria of good surgical practice as described are fulfilled, with standardized
lesions, controlled duration of surgery, and strict control of temperature and absence of
desiccation. It should moreover be realized that the laparoscopic mouse model, is a model
for three distinct pneumoperitoneum conditions: normoxia, hypoxia and hyperoxia. The first
normoxia model with addition of 4% oxygen intends to minimize any peritoneal damage
except for the lesions inflicted to induce adhesions on uterus and abdominal walls. Thus,
adhesions would form according to the classic model, with little or no effect on the
peritoneal cavity. The second model is the hypoxia model, since adhesions are enhanced by
CO2 pneumoperitoneum induced mesothelial hypoxia. In the third model, called the
hyperoxia model, 12% of oxygen was added to the CO2 pneumoperitoneum, a concentration
known to enhance adhesions probably by cell damage by reactive oxygen species.
The use of dexamethasone decreased adhesions by some 30% in the hypoxia model 52, by
60% in the hyperoxia model, and by some 76% in the normoxia model when it is combined
with low temperature. ROS scavengers decreased adhesions by 10 to 15% in the hypoxia and
hyperoxia model, an effect too small to be demonstrated in the normoxia model, with fewer
adhesions to start with. Calcium channel blockers decrease adhesion formation by some 35%
of inhibition in both hypoxia and hyperoxia models, and around 58% in the normoxia model
when is combined with low temperature; recombinant Plasminogen Activator (rPA) decrease
adhesion formation by 40% in the hypoxia and normoxia models whereas less inhibition,
around 17%, was observed in the hyperoxia model. Ringers lactate as a flotation agent was
marginally but significantly effective56. The effects of other flotation agents as
carboxymethylcellulose (CMC) and Hyskon were marginal (and surfactants as phospholipids
gave some 35% of inhibition in the hypoxia and hyperoxia models and 58% in the normoxia
19
model when is combined with low temperature52.Icodextrin, (Adept, a 4% α 1-4 glucose
polymer) unfortunately could not be evaluated since it degrades enzymatically in mice.
Barriers as Hyalobarrier gel, spraygel and Intercoat were highly effective in all models with a
reduction of adhesions between 58 and 90%.
Prevention of angiogenesis also reduces adhesion formation, as demonstrated in PlGF
knockout mice and by the administration of anti VEGF and anti PlGF monoclonal
antibodies40;55.
The transplantation of cultured mesothelial cells into the peritoneal cavity also is effective in
decreasing adhesion formation and in remodeling the area of mesothelial denudation as
described earlier (cfr. supra).
Translation of these findings to the human is not evident, as mechanisms may alter between
species. All the principles of good surgical practice were implemented in microsurgery, but
not in laparoscopic surgery and even then are adjuvant measurements necessary to further
reduce adhesion formation. The last decade many products have been tested in the
prevention of adhesions in human.
Flotation agents as Ringers lactate are marginally effective and its efficacy has not been
proven. Adept® (Icodextrin, Baxter, Deerfield, Illinois, USA) a macromolecular sugar with a
high retention time in the peritoneal cavity has shown some evidence of adhesion
prevention in a clinical trial versus ringers lactate56. There was a significant difference of
9.8% between Adept (49%) and ringers lactate (38%) in prevention of de novo and adhesion
reformation. A major advantage is the safety and absence of side effects, which were well
established since extensively used for peritoneal dialysis. The strength of the available
evidence demonstrating efficacy was considered not very solid in a Cochrane review57.
Interceed® (Johnson & Johnson, Gynecare, Somerville, New Jersey) is a biodegradable
oxidized regenerated cellulose sheet to be used in open and laparoscopic surgery. The use in
laparoscopy however is not practical and complete hemostasis is necessary to have the
desired effect in adhesion formation reduction. A significant effect of 32% reduction in
incidence of adhesions was shown compared to good surgical practice alone58.
Seprafilm® (Genzyme Corporation, Cambridge, Massachusetts) is a hyaluronic acid film
degraded over a 2 tot7 day period. It does not require complete hemostasis, but is also
difficult to use during laparoscopic surgery. It was used in only one trial for laparoscopic
myomectomy to assess the incidence, severity, extent, and area of uterine adhesions after
20
myomectomy59. Seprafilm significantly reduced the incidence postoperative uterine
adhesions: mean number of sites adherent tot the uterus = 4.98 vs. 7.88 (p<0.0001).
Additionally, Seprafilm treatment was not associated with an increase in postoperative
complications.
Since Intergel (0.5% ferric hyaluronate gel) has been withdrawn from the market, only
Hyalobarrier gel (Auto-cross linked hyaluronic acid gel), Spraygel (Polyethylenglycol) and
Intercoat/Oxiplex remain available as gels for clinical use. Overall efficacy appears to be
similar for all 3 products. A comparison between these 3 gels can unfortunately not be made
since comparative trials do not exist. Also the strength of the available evidence varies and a
Cochrane review of hyaluronic acid and Spraygel concluded that only for hyaluronic acid the
evidence was solid57. Most important is that for none of these products efficacy has been
proven for endpoints that really matter, i.e. pain, infertility, and bowel obstruction or
reoperation rate. We moreover should realize that large RCT trials were needed because of
the high intra-individual variability, and that in these trials the surgical interventions were
limited to rather simple and straightforward interventions as cystectomy and myomectomy.
In addition, these trials have been performed during interventions performed by laparotomy
or by laparoscopy under conditions of CO2 pneumoperitoneum enhanced adhesion
formation and slight desiccation. It therefore is still unclear to what extent the available
results of efficacy can be extrapolated to more severe or other types of surgery, whether
highly variable efficacy in adhesions and in prevention, is patient or intervention or surgeon
dependent and whether in the human the effect will be additive to good surgical practice
and conditioning of the peritoneal cavity.
Studies on anti-adhesion products should always include second-look laparoscopy to assess
incidence, extent and severity of adhesions as this may represent the true effect of the
method used. In order to address and compare adhesions a reliable and validated scale
should be implemented.
21
D. Important clinical factors related to the pneumoperitoneum
I. CO2 absorption during laparoscopy
Cardiovascular stimulation with tachycardia, arrhythmias and hypertension occur when
intra-abdominal pressure (IAP) exceeds 12-14 mm Hg; these effects are not clinically
relevant in ASA I and II patients but can compromise ASA III-IV patients60;61. The venous
blood return is reduced by the elevated IAP and intermittent pneumatic compression is
recommended to reduce the venous stasis. IAP must be kept low to limit impaired organ
perfusion. A sudden drop in blood pressure and in end-tidal CO2 concentration during gas
insufflation is a sign of gas embolism.
CO2 pneumoperitoneum also causes hypercapnia and respiratory acidosis. During
laparoscopy, monitoring of end-tidal CO2 concentration is mandatory. Normocapnia should
be maintained by increasing minute volume and frequency of ventilation and by changing
ventilatory pattern (Intermittent positive pressure ventilation (IPPV) to pressure controlled
ventilation (PCV)). IAP must be kept as low as possible to reduce CO2 absorption and to
protect the microcirculation of the peritoneal surface. Acidosis may cause the following
effects: O2Hb dissociation curve shift to the right, depression of myocardial contractility,
decreased
sensitivity
to
catecholamine,
venoconstriction
of
peripheral
veins,
vasoconstriction of pulmonary arteries, and hyperkalemia by extracellular shift of potassium.
In patients with limited pulmonary reserves, CO2 PP carries an increased risk of CO2
retention, also after the operation.
When ventilation is kept constant in rabbits, as was shown by Mynbaev et al.62, CO2pneumoperitoneum profoundly affected blood gases and acid base homeostasis i.e.
increasing pCO2, HCO3 (P < 0.001) and lactate concentrations (P < 0.05) and decreasing pH,
actual base excess and standard bicarbonate (P < 0.001), resulting in metabolic hypoxemia
with desaturation, lower pO2 (P < 0.001) and O2Hb (P < 0.05). These effects were more
pronounced with higher insufflation pressure and suboptimal ventilation
CO2 absorption from the pneumoperitoneum increases over time. Rabbits eventually died as
acidosis increased.
22
Acidosis in rabbits was prevented by adding 4% of oxygen to the CO2 pneumoperitoneum63.
This increase in CO2 absorption was equally decreased by adding oxygen and it is reasonable
to assume that the mesothelial hypoxia causes a trauma exposing directly extra cellular
matrix (ECM). Adding 4% of oxygen results in a partial oxygen pressure of 30 mmHg (4% of
760 atmospheric pressure + 15 mmHg insufflation pressure) which is within the normal
physiologic range of 20-40 mmHg for peripheral tissues. This should prevent this mesothelial
hypoxia, thus preventing the exposure of ECM, thus preventing the increased CO 2
absorption.
II. Nitrous oxide pneumoperitoneum in laparoscopy
Considering the cardiovascular and other side effects of CO2, other gases have been explored
for pneumoperitoneum especially, helium and N2O. The former is an inert gas but has a low
solubility in water. The latter has a solubility and lung exchange capacity similar to CO 2, and
thus is inherently safe.
Nitrous oxide (N2O) has long been used in laparoscopy for sterilizations or diagnostic
procedures as it had beneficial effects on pain during and after surgery compared to carbon
dioxide. The use of this gas was always debated as it displays a risk of explosion with
concentrations above 30% in combination with a combustible gas such as methane, i.e.
released during colonic surgery and an electrical charge.
Nitrous oxide shares several advantageous properties compared to CO2: it is an inexpensive
gas, it is rapidly eliminated, and it has diffusion and solubility quotients similar to CO 2. It also
has anesthetic and analgesic properties, without the cardiopulmonary side effects of CO264.
Several clinical and experimental studies demonstrated the safety and the advantages of
N2O pneumoperitoneum65.
In a clinical trial Tsereteli et al. demonstrated that the use of nitrous oxide compared to
carbon dioxide for pneumoperitoneum significantly decreased post-operative pain and
reduced the end-tidal CO2 during laparoscopy66. As investigated by Wont et al. carbon
dioxide caused a slight worsening of the parietal peritoneal acidosis at higher intraabdominal pressure (12-15 mmHg), whereas, nitrous oxide, helium and lifting of the
abdomen did not cause parietal peritoneal acidosis67. Again the reduction in acidification of
23
the peritoneum by the use of nitrous oxide can be the cause for the beneficial effects as was
demonstrated by Tsereteli et al. but is still unknown.
III. Tumor implantation in laparoscopy
Laparoscopic surgery in patients with intra-abdominal cancer is controversial since in animal
experiments, laparoscopy had a stimulating effect on tumor growth compared to
laparotomy68. Survival in women undergoing laparoscopy for ovarian cancer, however, was
not impaired69.
The positive effect of the CO2 pneumoperitoneum in animal studies on tumor implantation
was mainly attributed to the influence of the CO2 pneumoperitoneum on the dispersion of
free floating tumor cells. The effect of the carbon dioxide on tumor implantation and growth
was higher than other gasses (helium, nitrous oxide, xenon or gasless laparoscopy)70;71. This
detrimental effect therefore was attributed to the CO2 pneumoperitoneum, either as a
direct effect on the peritoneum or as an indirect effect on macrophage function72.
As homeostasis is the single most important factor to maintain the integrity of the
mesothelial lining, we demonstrated in previous experiments that the addition of 3% oxygen
to the CO2 pneumoperitoneum did indeed decrease adhesions73. Carbon dioxide damages
the mesothelial cell layer with direct exposure of the basal lamina as evidenced by scanning
electron microscopy. This exposure of extracellular matrix proteins such as laminin,
fibronectin and vitronectin to the tumor cell surface was suggested to increase tumor cell
adherence74. Since the cascade of events following pneumoperitoneum (i.e. mesothelial
damage and peritoneal healing leading to adhesion formation) might also be important in
implantation and growth of tumor cells75, one can anticipate the effect of a
pneumoperitoneum with pure CO2 and of adding oxygen to the CO2 pneumoperitoneum
upon tumor implantation in our laparoscopic mouse model for adhesion formation. If any
effect in the human by the addition of 4% of oxygen to the CO2 pneumoperitoneum on
incidence of port site metastases, by preventing tumor cell hypoxia caused by CO2
pneumoperitoneum remains to be answered.
Port-site metastases (PSM) are a frequent, but clinically not very important complication of
laparoscopy for malignant conditions76. PSM especially occur after laparoscopy in advanced
24
ovarian cancer with an incidence of 17% PSM. The risk of clinical PSM was independent of
the type of tumor, the tumor grade and the presence of ascites. Different mechanisms have
been suggested, such as direct wound contamination during extraction of the malignant
specimen through the laparoscopic port, aerosolization of exfoliated tumor cells, gas leaks
(known as the chimney effect) and a lowered immune response due to the carbon
dioxide70;77;78. Anyway, the concern remains that laparoscopic surgery, and more specifically
the CO2 pneumoperitoneum, might be a predisposing factor for PSM.
IV. Post-operative pain and inflammation in laparoscopy
Postoperative somatic pain is widely believed to be caused by the surgical injury of nerve
fibers and by the subsequent inflammatory reaction, a necessary mediator for healing of
tissues. Pain after laparoscopy is believed to be caused for 50% by the incision of the surgical
wounds, for about 30% by the pneumoperitoneum (PP) and for another 20% by the
operation wound79;80. This concept anyway has lead to the relatively widespread use of local
anesthesia of the skin before making an incision for laparoscopy. Visceral pain is known to be
very different from somatic pain. Visceral pain is indeed more mediated by stretching than
by injury, while over 80% of the visceral nociceptors are sleeping. Following inflammation,
the dormant nociceptors are recruited and the threshold is decreased leading to a much
increased firing activity to any stimulus including bowel movements81. The speed of
recruitment and activation of dormant nociceptors, however, has not been investigated in
detail. It equally is unclear what the exact time courses of pain are over the first 48 hours
after surgery. Given the difficulty to measure pain and the fact that most estimations are
indirect, this is not surprising.
It is not clear what the exact mechanism of pain is after laparoscopic surgery and why pain is
less when a similar type of surgery is performed by laparoscopy than performed by
laparotomy, as demonstrated for hysterectomy82 or cholecystectomy83;84. It is unclear
whether the abdominal incision itself is sufficient as an explanation, while the minimal
invasiveness of surgery is more a concept than a proven model to reduce pain. Anyway, after
laparoscopic surgery the systemic immune response is less pronounced in comparison with
open surgery as estimated by levels and duration of postoperative C-reactive protein (CRP)
25
and interleukin-637. The exact contribution of each component to pain after laparoscopic
surgery and how this fits with the known mechanisms of visceral pain mediation remains
unclear.
Without a surgical injury adhesions are not likely to form as we already demonstrated in our
laparoscopic mouse model41. Quantitatively however, the effects of acute inflammation of
the entire pelvic cavity were some 20 times more important. This acute inflammation of the
entire pelvic cavity was shown to be caused by duration and severity of mesothelial hypoxia
during CO2 pneumoperitoneum, by reactive oxygen species (ROS) and by even gentle
manipulation of bowels38. Each of these factors results in a retraction and bulging of the
mesothelial cells in the entire peritoneal cavity and an acute inflammatory reaction. We in
addition demonstrated that this peritoneal trauma could be decreased/prevented by adding
3% of oxygen to the CO2 pneumoperitoneum, i.e. a local physiologic partial oxygen pressure
of some 22 mm Hg (0.03*760), thus resulting in a decreased postoperative adhesion
formation73.
We therefore investigated whether the addition of 4% of oxygen to the CO2
pneumoperitoneum could decrease the peritoneal acute inflammatory reaction and
postoperative pain in women undergoing a promontofixation.
E. Aims of the thesis
To understand the aims of this thesis it is important to know the historical perspective in
which they were formed. From 1996 up to now, the concept of full conditioning was
developed through different steps. Roger Molinas and Mercedes Binda developed a
laparoscopic mouse model and looked at different factors influencing adhesion formation.
Hypoxia was recognized as an important factor and addition of 4% of oxygen was proven to
be the optimal concentration during laparoscopy. From this, the first translational trials in
the human were designed. Further research investigated desiccation and temperature as
important factors in the prevention of adhesion formation, but translation was only possible
through a joint venture with eSaturnustm. During the development of the equipment to
control for desiccation and temperature, the addition of nitrous oxide was investigated upon
by Roberta Corona and Karina Mailova in our laparoscopic mouse model. The concept of full
26
conditioning was further completed with the addition of dexamethasone and rinsing with
heparinized solution. Full conditioning and consequences on inflammation and adhesions
was investigated in parallel in our laparoscopic mouse model and in the human.
Additional research in mice on tumor implantation and transplantation of mesothelial cells
was done.
More specific hypotheses and general aims of this thesis were:
1/ Addition of 4% of oxygen to the pneumoperitoneum will prevent mesothelial hypoxia and
prevent retraction of the mesothelial cells and exposure of the extracellular matrix
2/ Addition of 4% of oxygen to the pneumoperitoneum will prevent mesothelial hypoxia and
will decrease CO2 absorption
3/ Addition of 4% of oxygen to the pneumoperitoneum will prevent mesothelial hypoxia and
will decrease tumor growth and tumor implantation in laparoscopic port sites
4/ Addition of 4% of oxygen to the pneumoperitoneum will prevent mesothelial hypoxia and
decrease post-operative inflammation and pain
5/ Full conditioning of the pneumoperitoneum will prevent mesothelial damage and will
decrease CO2 absorption
6/ Full conditioning of the pneumoperitoneum will prevent mesothelial damage and will
decrease post-operative inflammation and pain
7/ Full conditioning of the pneumoperitoneum will prevent mesothelial damage and will
decrease adhesion formation
8/ Full conditioning of the pneumoperitoneum will prevent mesothelial damage and
decrease speed of absorption of fluid (Ringer lactate or Adept®)
9/ Autologous transplantation of mesothelial cells after laparoscopy will decrease adhesion
formation
27
28
Chapter 2
General materials and methods
A. The laparoscopic mouse model
After having used rabbits for studying adhesion formation we progressively developed the
mouse laparoscopic model. This model actually is strictly standardized. The following are
important aspects: Balb-c mice are an inbred strain (and low variability) with genetically
important adhesion formation; anaesthesia, ventilation, temperature, timing of events,
surgical lesions and surgical experience. Adhesions can be scored after 7 days which
facilitates planning. The model permits 6-8 mice to be operated / day.
Each animal model has advantages and limitations. The main disadvantage of mice is the
size. In addition, the thickness of the entire abdominal wall is equivalent to the first muscle
layer in rats and the same relationship exists between rats and rabbits. The ratio of
peritoneal surface area / body weight is higher in smaller than in larger animals, being 0.195
m2/kg in mice, 0.115 m2/kg in rats, 0.084 m2/kg in rabbit and 0.026 m2/kg in humans. The
volume of peritoneal fluid is disproportional higher in smaller than in larger animals being
18.1 ml/kg in mice, 10.7 ml/kg in rats, 7.7 ml/kg in rabbit and 2.4 ml/kg in humans 85.
The mouse model has advantages: they are inexpensive and easy to handle. Several well
characterized types are available, such as inbred strains, genetically manipulated mice, e.g.
mice non-expressing or over-expressing specific genes, mice with an altered immune system,
e.g. nude and SCID mice. Moreover, many specific assays and monoclonal antibodies exist as
is the case for Balb/c mice.
Also the genetic background has an influence in adhesion formation. Some mouse strains
developed more adhesions than others and, in addition, outbred strains have more
variability in the results than inbred strains86, therefore, to have the maximum of adhesions
with less variability 9-10 weeks-old female BALB/c mice inbred strain of 18 to 24g were
used. As Balb/c mice are also not too expensive, it is was selected as the preferred animal for
our experiments.
29
In our laboratory a mouse model was developed in which many parameters were modulated
and/or monitored: conditions of the pneumoperitoneum (duration, pressure, type of gas,
humidification and temperature), body temperature and ventilation. All these parameters
can have an influence in adhesion development and should be controlled during the studies.
Exact timing was found to be crucial in order to standardize the slight hypothermia
associated with anesthesia54. After injection of pentobarbital 0.08 mg/g pentobarbital i.p.
(Nembutal, Sanofi Sante Animale, Brussels, Belgium) at time zero (T0) animals were put in a
conditioned room at 37°C. Further animal preparation, i.e. shaving, placement on the table
in the supine position, intubation and ventilation started exactly after 10 min (T10). Animals
were intubated with a 20-gauge catheter and ventilated with a mechanical ventilator
(Mouse Ventilator MiniVent, Type 845, Hugo Sachs Elektronik-Harvard Apparatus GmbH,
March-Hugstetten, Germany) using humidified air with a tidal volume of 250 µl at 160
strokes/min. Ventilation finished at T80, i.e. after a pneumoperitoneum of 60 minutes.
At exactly T20, CO2 pneumoperitoneum was induced using the Thermoflator plus® (Karl
Storz, Tüttlingen, Germany) through a 2 mm endoscope with a 3.3 mm external sheath for
insufflation (Karl Storz, Tüttlingen Germany) introduced into the abdominal cavity through a
midline incision caudal to the xyphoid process appendix. The incision was closed gas tight
around the endoscope in order to avoid leakage and thus higher flow rates of insufflation
gas. The insufflation pressure was set at 15 mmHg as insufflation pressure increased
adhesions as demonstrated in rabbits45;87 and in mice41;73. We decided to use this maximum
pressure in order to observe a maximum effect of ischemia/hypoxia and since adhesions are
pressure dependent. In addition, a pneumoperitoneum with a pressure of 15 mm Hg could
be relevant since it can mimic the conditions that happen in surgery on humans. For gas
humidification, the Storz Humidifier 204320 33 (Karl Storz, Tuttlingen, Germany) was used,
because when non-humidified gas is used to induce the pneumoperitoneum, desiccation
occurs and adhesions increase39. In addition, desiccation is associated with cooling. The CO2
pneumoperitoneum was maintained for 60 minutes until T80 .
Temperature of the mice was strictly controlled at 37°C using a closed chamber maintained
at 37°C (heated air, WarmTouch, Patient Warming System model 5700, Mallinckrodt
Medical, Hazelwood, MO). All equipment and mice were placed in this chamber, thus
keeping them at a constant 37°C throughout the experiment. The temperature in the
chamber was measured with a Testo 645 device (Testo N.V./S.A., Lenzkirch, Germany),
30
whereas the temperature of the animal was measured via the rectum with a Hewlett
Packard 78353A device (Hewlett Packard, Böblingen, Germany).
After the establishment of the pneumoperitoneum, two 14-gauge catheters (Insyte-W,
Vialon, Becton Dickinson, Madrid, Spain) were inserted under laparoscopic vision.
Standardized 10 mm x 1.6 mm lesions were performed in the antimesenteric border of both
right and left uterine horns and in both the right and left pelvic side walls with bipolar
coagulation (20W, standard coagulation mode, Autocon 350, Karl Storz, Tüttlingen,
Germany). (Figure 2)
Figure 2: the laparoscopic mouse model
Since adhesion scores on day 7 were equal to adhesion scores on day 14 or later, scores on
day 7 were taken. Adhesions were scored quantitatively (proportion and total) and
qualitatively (extent, type, tenacity) blindly by laparotomy under a stereomicroscope. The
qualitative scoring system assessed: extent (0, no adhesions; 1, 1–25%; 2, 26–50%; 3, 51–
75%; 4, 76–100% of the injured surface involved, respectively), type (0, no adhesions; 1,
filmy; 2, dense; 3, capillaries present), tenacity (0, no adhesions; 1, easily fall apart; 2,
require traction; 3, require sharp dissection) and total (extent + type + tenacity). The
quantitative scoring system assessed the proportion of the lesions covered by adhesions
31
using the following formula: (sum of the length of the individual attachments/length of the
lesion) ×100. The results are presented as the average of the adhesions formed at the four
individual sites (right and left visceral and parietal peritoneum), which were scored
individually. The entire abdominal cavity was visualized using a xyphopubic midline and a
bilateral subcostal incision. After the evaluation of ports sites and viscera (omentum, large
and small bowels) for de novo adhesions, the fat tissue surrounding the uterus was carefully
removed. The length of the visceral and parietal lesions and adhesions were measured.
Adhesions, when present, were lysed to evaluate their type and tenacity. The terminology of
Pouly was used1, describing de novo adhesion formation for the adhesions formed at nonsurgical sites, adhesion formation for adhesions formed at the surgical site and adhesion
reformation for adhesions formed after the lysis of previous adhesions.
B. Clinical trials
All patients for the clinical trials were recruited at the University Hospital Leuven, campus
Gasthuisberg. Written and informed consent was obtained for all.
Inclusion criteria always consisted of women undergoing gynaecologic laparoscopic surgery
for at least 60 minutes of planned laparoscopy time and signed informed consent.
Exclusion criteria always mentioned: pregnancy and women with a pre-existing condition
increasing the risk of surgery such as clotting disorders, heart or lung impairment. All
conditions that might interfere with inflammatory parameters such as immunodeficiency,
chronic inflammatory disease as Crohn’s disease were also exclusion criteria except for the
trial on port-site metastasis and the first trial on CO2 absorption.
Approval of the ethical committee from the University Hospital Leuven was obtained for all
clinical trials.
Clinical trials were registered through clinicaltrials.gov:
NCT00678366:
Evaluation
of
Adding
Small
Amounts
of
Oxygen
to
the
CO2
Pneumoperitoneum Upon Pain and Inflammation
NCT01344499: Clearance Rate of Peritoneal Fluid After Full Conditioning Pneumoperitoneum
NCT01340989: CO2 Absorption During Laparoscopy
NCT01344486: Peritoneal Cavity Conditioning Decreases Pain, Inflammation and Adhesions
32
I. Adding 4% oxygen to the pneumoperitoneum
For the clinical trials where 4% of oxygen was added to the carbon dioxide
pneumoperitoneum the Thermoflator plus® (Karl Storz AG, Tüttlingen, Germany) was used
for insufflation. This insufflator allowed a manual regulation of the amount of oxygen added
to the carbon dioxide pneumoperitoneum from 1 to 12%.
The mixture of gas is patented through KUL research and development.
II. Full conditioning of the peritoneal cavity
In the trials where full conditioning was used, a premixed bottle of 86% carbon dioxide +
10% nitrous oxide + 4% oxygen was used. All bottles were officially certified to have the
correct mixture. The gas had the same composition at the beginning and at the end of the
bottle, so no different composition of the gas occurred over time (Ijsfabriek Strombeek).
Full conditioning further existed in insufflation temperature regulation at 32°C and 100%
humidification through a modified Fisher and Paykel (F&P) humidifying system (Type: MR860
AEA, SN: 070514000016, Fisher & Paykel Healthcare®, Auckland, New Zealand). The F&P
humidifier blows gas over a heated water chamber. In order to avoid condensation, the
tubing delivering the gas is heated to over 40°C with a heating wire. In order to permit
higher flow rates, the luer lock limiting flow rates to 7.8 L/min at 15 mm Hg insufflation
pressure, was removed for our experiments. In the F&P humidifier, modified by eSaturnus®,
the heating of the chamber and the heating of the tubing was continuously adapted by an
electronic feedback loop in order to maintain at the end of the tubing 100% relative
humidity (RH) at a preset temperature between 25°C and 36°C and this at any flow rate
between 0.5 and 30 liters/min. The flow rate, relative humidity and temperature of the gas
at the end of the insufflation tubing and of the gas outflowing from the peritoneal cavity or
from the box used for the experiments, were measured with a sensor for temperature and
relative humidity (model SHT75, Sensirion, Switzerland).
Both composition of mixture of gas and cooling by third means are patented through KUL
research and development.
33
Figure 3: set-up for full conditioning
The set-up of the system was tested to ascertain the control of temperature and
humidification. (Figure 3)
First in vitro experiments using a closed polystyrene box imitating a clinical setting by placing
heated water at 37°C inside the box were carried out. From these experiments the setting
for the steering of the humidification and heating in a clinical setting were determined.
Second we carried out an observational clinical trial with different flow rates (2.5, 5, 7.5, 10
and 15 liters per minute) and RH and temperature in and out were measured. (Figure 4)
These data permitted us to use the insufflator at different flow rates and to maintain 100%
RH at insufflation with an inflow temperature set at 32°C. In vitro and in vivo data were
similar.
34
Figure 4: relative humidiy and temperature with different flow rates
The system could correct for any cooling inside the abdominal cavity as well as for heating by
coagulation and maintain a 32°C inside the abdominal cavity. A sprinkler with normal saline
was inserted into the abdominal cavity to correct for any loss of water and thus loss of
energy by the body. (Figure 5)
Figure 5: Loss of water without and with humidifier
The use of humidified, mixed (86% CO2 + 4%O2 + 10% N2O) gas at 32°C with the use of the
sprinkler is referred to as “full conditioning”.
35
An overview of the surgical procedures in the clinical trials using full conditioning is shown in
table 1.
Type of laparoscopic
surgery
CO2 absorption
Pain inflammation
Adhesions
Fluid absorption
Deep endometriosis
+
+
+
-
Total hysterectomy
+
+
-
-
Myomectomy
+
+
-
-
Colpopexia
+
+
-
-
Adhesiolysis
+
+
-
-
Subtotal hysterectomy
-
+
-
+
Table 1: overview of different trials using full conditioning
C. Statistics
Statistical evaluation was done with Prism or with the SAS system, using paired and unpaired
t-tests for normally distributed populations or Wilcoxon, (proc n-par1way) for non normally
distributed populations. Correlations were evaluable by the Spearman and or Pearson test.
Two way analysis of variance was done with two way ANOVA or proc GLM, for normally and
non normally distributed populations respectively.
For animal experiments, a factorial design was generally used, but also for the trial on fluid
absorption. Indeed a strictly two x two factorial design (latin cross) generates almost similar
significances and power for two variables with in addition interaction, for almost half the
number of mice as would have been required when each factor would have been
investigated separately.
36
Chapter 3
Results
A. Animal experiments
I. Establishing a learning curve
Corona R, Verguts J, Binda M, Molinas R, Schonman R, Koninckx P. The impact of the learning
curve upon adhesion formation in a laparoscopic mouse model, Fertil Steril, accepted
AIM: to assess the effect of training assessed by consecutive surgeries, upon operating time
and upon the extent and variability of post operative adhesion formation.
MATERIALS & METHODS: The operating time was measured from T20, i.e. from the beginning
of surgery, exactly 20 minutes after induction of anesthesia, until the end of the procedure
with the removal of catheters for instrumentation and port sites closure. Postoperative
formation of adhesions was evaluated blindly by an independent experienced observer 7
days later. Moreover, the codes of intervention order were broken only at the end of the
training experiment. The study was performed as a prospective randomized (for scoring) trial
initially in Swiss mice and later in Balb/c mice. Each trainee performed sequentially 80
interventions to induce adhesion formations. All trainees were inexperienced for the
laparoscopic procedure in a mouse model, but some of them were experienced
gynecologists after their training in gynecology whereas others were inexperienced at the
end of medical school.
All experiments were performed using block randomization by days. Therefore, a block of
animals comprising one animal of each strain, one Balb/c and one Swiss mouse, were always
operated in a single session the same day to avoid day-to-day variability. In addition, within a
block, experiments were performed in random order. Before surgical procedure, initial
temperature and weight were measured.
37
Statistical analyses were performed using GraphPad Prism version 4 (GraphPad Software
Inc., San Diego CA). Multifactorial analyses were achieved with GLM and Logistic procedures
using the SAS System (SAS Institute, Cary, NC, USA).
RESULTS: Experienced (gynecologist) and inexperienced (undergraduate) surgeons had to
operate each 80 mice (40 blocks of 1 Balb/c and 1 swiss mouse) before being permitted to
do experiments. This was derived from clinical impression, showing that initially duration of
surgery, adhesions and variability was much higher. With training, assessed by consecutive
surgeries, duration of surgery decreased exponentially in both groups, demonstrating a clear
learning curve that can be described as a two-phase exponential decay model as shown in
figure 1. (non-experienced: R=0.72; experienced: R=0.73) (Figure 6). In Balb/c mice the real
and calculated operating times for non-experienced surgeons decreased from 19 ± 6 min
and 22 ± 4 min for the first procedures to 7 ± 1 min and 7 ± 1 min for the last procedures
(P=0.0001; P=0.0001, respectively). For experienced surgeons time decreased from 8 ± 1 min
and 10 ± 3 min for the first ten procedures to 4 ± 0.3 min and 4 ± 0.03 min for the last ten
Operation time (minutes)
procedures.
Non Experienced
Experienced
Figure 6: Duration of surgery during training: Learning curve expressed as duration of the surgery vs
number of consecutive procedures
With experience also adhesion formation decreased (Figure 7) for both experienced and
inexperienced surgeons. Adhesion formation was lower with the consecutive number of
surgeries (proportion: p<0.01; total: p<0.01), for surgeries of shorter operating time
38
(proportion: p< 0.02;
total: p< 0.04), and among experienced surgeons (proportion:
p<0.0001; total: p< 0.04).
Non Experienced
Experienced
Figure 7: Adhesion formation during learning curve: Adhesion formation expressed as proportion vs
number of consecutive blocks in both experienced and non experienced surgeons.
It was decided to arbitrarily group the blocks of animals in 4 groups of ten blocks each (G1,
block 1-10; G2, block 11-20; G3, block 21-30; G4, block 31-40). With this grouping, the interanimal variability for operating time and for adhesion formation was assessed by the
coefficient of variation (CV). The CV of operating time among non-experienced surgeons was
41%, 35%, 25% and 20% for Balb/c mice for the first, second, third and fourth group,
respectively; among experienced surgeons the CV was 45%, 24%, 20% and 22% for Balb/c
mice for the first, second, third and fourth group. Taken all surgeons together the CV of the
proportion of adhesions was 62%, 48%, 54% and 38% for Balb/c mice for the first, second,
third and fourth group, respectively.
Both operating time and its CV decreased with the number of surgeries (p<0.0001 for both),
whereas they were not affected by mouse strain (p=NS).
These results suggest that experience and thus decreased manipulation and operating time
will decrease mesothelial damage as this damage may be inflicted by even minor
manipulation of intra-abdominal organs.
39
II. Mesohelial cells in prevention of adhesions
Verguts J, Coosemans A, Corona R, Praet M, Mailova K and Koninckx P. Intraperitoneal
injection of cultured mesothelial cells decrease CO2 pneumoperitoneum-enhanced adhesions
in a laparoscopic mouse model. Gyn Surg. 2011(8)-addendum
AIM: Autologous transplantation of mesothelial cells after laparoscopy will decrease
adhesion formation
MATERIALS & METHODS: Mesothelial cells were obtained from 9-16 week old inbred female
Balb/c mice. Following anesthesia with intradermal pentobarbital (Nembutal, Sanofi Sante
Animale, Brussels, Belgium; 0.06 mg/g) the abdomen was shaved, the animal was secured to
the table in supine position and the skin disinfected with a 50% alcohol solution. A 14-gauge
catheter (Insyte, Vialon, Becton Dickinson) was inserted into the abdomen at the umbilicus
and fixed leak-proof with a Prolene 5/0 purse suture.
The cells were collected via a 2-step procedure per mouse. Firstly, 10ml phosphate buffered
saline pH 7.3 (DPBS) was injected, of which at least 8 ml could be re-aspirated. This first set
of ‘free floating cells’ was centrifuged at 200G for ten minutes and the pellet was then
suspended in culture medium (RPMI 1640 (Lonza, BioWhittaker) supplemented with 15%
fetal calf serum (FCS; CSL UK Ltd, Andover, UK), 4 mM L-glutamine (Gibco, Paisley, UK), 0.4
mg/ml hydrocortisone (Sigma Aldrich, Poole, UK) and antibiotics: penicillin, 120mg/l,
amphothericine (2,5mg/l) and gentamicine (4mg/l) as used by Foley-Comer et al.85
Secondly, 10ml of DPBS with 0.25% Trypsin and 1 mM EDTA4Na at 37°C was injected
through the same catheter and the mouse was softly shaken. After ten minutes the instilled
fluid was re-aspirated. Next, the abdominal cavity was washed with 5ml culture medium; to
finish, the abdominal cavity was opened and gently rinsed 8 times with 2 ml culture medium.
All mesothelial cells obtained via this second procedure were collected together, centrifuged
for 10 minutes at 200g and re-suspended in culture medium. Each time two mice had
undergone the procedure, cells of those mice were pooled, keeping cells of step 1 and 2
separately. Cells were then put in an incubator at 37°C with 5% CO2. If cells were confluent,
which was normally the case after 1 week, cells were split at a ratio 1:2, after having them
40
detached with Trypsin-EDTA. After 3 weeks, cells were detached and counted for further use
(pilot: 400.000 cells, dose-response: 400.000 cells, 133.000 cells and 44.000 cells).
To ascertain the growth of mesothelial cells, cells from 3 weeks old cultures were seeded on
slides. After 2 days of culture, slides were fixed and endogenous peroxidase activity was
blocked by 0.5 % H2O2 in methanol. Cells were evaluated for intercellular connections and
for cytokeratin staining.
All mice received 4 standard bipolar lesions and 60 minutes of CO2 pneumoperitoneum as
described above. After all laparoscopic ports had been closed, 1ml of DPBS with or without
mesothelial cells, was injected intraperitoneally.
A first experiment was designed to prove that cultured mesothelial cells could prevent CO2
pneumoperitoneum enhanced adhesions. Mesothelial cells were collected in 4 mice, and
cultured in 8 flasks of 25cm². Two weeks later 2x106 cells were harvested. In order to
investigate the effect in 5 mice 400.000 cells per mouse were used. The control group which
only received DPBS consisted of 5 mice.
A second experiment was designed to obtain a dose response curve of adding different
amounts of cultured mouse mesothelial cells in adhesion reduction. Considering the effect of
400.000 cells from the first experiment, and taking into account the number of cells
available, we decided to inject per group of 5 mice 400.000 cells, 133.000 cells and 44.000
cells, i.e. a logarithmic decrease. The control group which only received DPBS consisted of 5
mice. The study was approved by the Institutional Review Animal Care Committee.
A third experiment was performed with 144.000 cells remaining from the second experiment
to evaluate, as a pilot experiment, whether these cultured mesothelial cells adhered
specifically to the lesion or randomly, over the entire peritoneum. The cultured mesothelial
cells were suspended in methylene blue (10mg/ml glucose solution) for two minutes,
centrifugated and re-suspended in 1ml of DPBS. The mesothelial cells were injected after a
surgical lesion and 60 minutes of CO2 pneumoperitoneum. Blue staining was evaluated after
two days using a stereomicroscope.
In a fourth experiment, designed to evaluate whether methylene blue injected
intraperitoneally did color the entire peritoneal layer or rather specifically the surgical lesion
and the retracted areas between the mesothelial cells following 60 minutes of CO2
pneumoperitoneum, 1ml of methylene blue was injected intraperitoneally either in a control
41
mouse without pneumoperitoneum, or in a mouse with a standard lesion and 60 minutes of
CO2 pneumoperitoneum. Both mice were evaluated after 2 days using a stereomicroscope.
RESULTS: Mesothelial derived from the mice were easily cultured and were initially bipolar
or multipolar in appearance but at confluence they adopted a polygonal configuration as
described by Yung et al.88 Viability of cells as estimated by tryptan blue exclusion was over
95%. The cells had intercellular connections and were positive for cytokeratin staining
confirming their mesothelial nature.
Cells obtained from the first washing did not grow, but remained viable as estimated by
tryptan blue exclusion after three weeks of culture. Their number, however, was too low for
subsequent experiments.
In the first experiment the injection of 400.000 cultured mesothelial cells caused a reduction
of the proportion of adhesion formation from 19% to 7% (p<0.046) and a reduction of the
total adhesion score from 3.1 (+/- 1.74) to 1.7 (+/-1.68) (NS, p<0.086).
Dose Response Curve
Total Adhaesion Score (mean/SD)
4.0
3.5
3.0
2.5
2.0
1.5
1.0
0.5
0.0
0
100000
200000
300000
Dose: number of cells
Figure 8: dose-response curve
42
400000
500000
The second experiment confirmed that mesothelial cells can reduce CO2 pneumoperitoneum
enhanced adhesion formation. With 44.000, 133.000 and 400.000 cells the proportion of
adhesions dropped from 13.5% to 11.5% (NS), 8% (P<0.055) and 5% (P<0.007) respectively.
The total adhesion score showed a similar decline being for the 44.000, 133.000 and 400.000
cells not significant, P=0.0093 and P=0.0025 respectively. (Figure 8) When adhesions are
plotted against the logarithm of the concentration of cells, the amount of cells required to
decrease adhesion formation by half was estimated at 100.000 cells.
Following injection of methylene blue stained mesothelial cells, after a standard lesion and
60 min of CO2 pneumoperitoneum, blue spots were visualised on the lesion site (picture 1)
while faint blue spots were found over the entire parietal peritoneum but not on the bowel.
In the control mouse however, without surgery nor pneumoperitoneum, no blue staining
was seen after injection of methylene blue stained mesothelial cells.
Picture 1: Following injection of methylene blue only a similar blue staining was found on the surgical
lesion and all over the parietal peritoneum after 60 minutes of pneumoperitoneum
43
III. Tumor implantation
Verguts J, Corona R, Binda M, Koninckx P. Adding 3% of oxygen to the CO2
pneumoperitoneum reduces tumor implantation in a laparoscopic mouse model. Clin Exp
Metastasis, submitted, 2011
AIM: Addition of 4% of oxygen to the pneumoperitoneum will prevent mesothelial hypoxia
and will decrease tumor growth
MATERIALS & METHODS: Mouse CT26 (colon adenocarcinoma) and RENCA (renal
adenocarcinoma) cell lines, syngeneic to Balb/c mice, were generously provided by Dr.
Markus Steinbauer from the University of Regensburg, Regensburg, Germany and Dr. Takumi
Takeuchi from the University of Tokyo, Japan respectively. Before use, cells were cultured in
RPMI-1640 medium supplemented with 10% FCS, 1% penicillin/streptomycin and 1% Lglutamine at 37°C in a humidified environment with 5% CO2. Cells were harvested,
suspended in culture medium and rinsed by centrifugation at 3000 RPM (1509g) for 10
minutes. Cell viability was assessed by the tryptan Blue exclusion test (which always
exceeded 95%) and counted in a Neubauer counting chamber. The final concentration was
diluted using culture medium to the desired concentration for each experiment. Cells were
injected intraperitoneally (i.p.) in 1.0 ml culture medium immediately after deflation of
pneumoperitoneum and closure of ports.
Animals were sacrificed by cervical dislocation. The abdominal cavity was exposed using a
xyphopubic midline incision and examined for tumor nodules under stereomicroscopic vision
x10. Nodules of more than 1 mm present in the abdominal wall and mesentery were scored
blindly by two independent investigators, the codes of groups and inoculations being broken
only at the end of the entire experiment.
All experiments were performed using block randomization by day. Therefore, a block of
animals comprising one animal of each group was always operated the same day, avoiding
day-to-day variability. Within a block, experiments were performed in random order, varying
each day. Laparoscopy was performed as described earlier (cfr supra), but no lesions on the
uterus or abdominal wall were made in this experiment.
44
Dose-response curve experiments with CT26 (“experiment A”) and RENCA cells
(“experiment B”).
These experiments were designed to estimate the optimal amounts of cells to be used in
further experiments by injecting increasing amount of tumor cells intraperitoneally. Animals
were anaesthetized and a variable amount of cells, suspended in 1 ml of medium, were
inoculated i.p. 2 cm caudal to the xyphoid. In experiment A and B, 63 mice (n=9 in each
group, seven groups) were injected with 103, 3x103, 104, 3x104, 105, 3x105 or 106 CT26 or
Renca cells (Groups: G1, G2, G3, G4, G5, G6 and G7 respectively). From each group, 3 mice
were examined for tumor growth after respectively 7, 14 and 21 days.
Experiments to evaluate the effect of pneumoperitoneum and of adding 3% of oxygen to
the pneumoperitoneum using CT26 and RENCA cell lines (“experiment C” and “experiment
D”) upon tumor implantation.
The mice of the control group (CG) were anaesthetized and ventilated only, whereas in mice
of Group 1 (G1) and Group 2 (G2) a pneumoperitoneum was maintained for 60 minutes with
CO2 + 3% O2 and with pure CO2 respectively.
In experiment C, 20 mice in each of the 3 experimental groups (CG, G1 and G2) (n=60) were
injected with 103, 3x104, 105 or 106 CT26 cells (5 mice/concentration/experimental group).
Mice inoculated with 105 and 106 cells were sacrificed and examined after 7 days and mice
inoculated with 3x103 and 3x104 cells were sacrificed and examined after 14 days.
In experiment D, 15 mice in each of the 3 experimental groups (CG, G1 and G2) (n=45) were
injected with 3x104 and 105 cells (5 mice/3x104 cell concentration/experimental group; and
10 mice/105 cell concentration/experimental group). Mice inoculated with 3x104 cells were
sacrificed and examined after 14 days whereas mice inoculated with 105 cells were sacrificed
and examined after 14 (n=15) and 21 days (n=15) respectively.
Experiments were performed in blocks. Each block comprised the three experimental groups
(CG, G1 and G2) with the 4 (CT26) or 3 (RENCA) cell concentrations i.e. 1 mouse/experimental
group/cell concentration. Cells were inoculated i.p. 2 cm caudal to the xyphoid immediately
after deflation and closure of port sites.
RESULTS: Experiments A and B: Following inoculation of exponentially increasing
concentrations of CT26 or RENCA cells, tumor implantation occurred mainly in the
mesentery, pancreas, subhepatic area, bladder, ovarian fat and abdominal wall. For both cell
45
types, the number of visible tumor nodules increased linear with increasing number of
inoculated cells (for CT26 cells after 7, 14 and 21 days: R2= 0.9283, R2= 0.8679 and R2=
0.4316 respectively; for RENCA cells after 14 and 21 days: R2 = 0.9795 and R2 = 0.9910
respectively). After 21 days, all animals with more than 104 injected cells had died. In mice
inoculated with RENCA cells and examined after 7 days, no tumor nodules were found,
whereas after 21 days tumor nodules were evident within mice injected with 10 3 cells.
(Figure 9)
Figure 9: Dose response curves for CT26 and RENCA
The size of tumor nodules increased with time and with the number of cells injected.
Experiments C. Effect of 60 min of pneumoperitoneum upon CT26 cells implantation. In
comparison with the control group, tumor implantation increased after one hour of pure
CO2 pneumoperitoneum for all doses used. This increase was found for total number of
tumor nodules (Figure 10) and for number of nodules in mesentery and abdominal wall
(Table 2) (3x103: Mesentery: P= .04, Abdominal wall: P= .04, Total: P= .04; 10 5: Mesentery:
P= .03, Abdominal wall: P= .03, Total: P= .03 and 10 6: Total: P= NS). In comparison with pure
CO2 pneumoperitoneum, tumor implantation decreased with the addition of 3% of oxygen
to the CO2 pneumoperitoneum for all doses used. This decrease was found for total number
of tumor nodules and for number of nodules in mesentery and abdominal wall (Table 2)
(3x103: Mesentery: P= NS, Abdominal wall: P= NS, Total: P= .05; 105: Mesentery: P= .NS,
Abdominal wall: P= .NS, Total: P= .04 and 106: Mesentery: P= NS, Abdominal wall: P= NS,
Total: P= NS). In comparison with the control group, tumor implantation increased after one
hour of CO2 pneumoperitoneum and 3% of oxygen for all doses used. This increase was
46
found for total number of tumor nodules (Figure 10) and for number of nodules in
mesentery and abdominal wall (Table 2) (3x103: Mesentery: P= .04; Abdominal wall: P= .04;
Total: P= .04; 105: Mesentery: P= .04, Abdominal wall: P= .04, Total: P= .04 and 106:
Mesentery: P= NS, Abdominal wall: P= NS, Total: P= NS).
Table 2: nodules per type of tumor and per site, a P vs. Group + 3%oxygen <.05; b P vs. Control <.05.
In the group injected with 3x103 cells, one mouse in G1 and G2 died 1 day before the
examination. The group injected with 3x104 cells had a high mortality rate which didn’t allow
any difference to appear, a total of 11 animals died after 12 days, 2 animals in CG, 4 animals
in G1 and 5 animals in G2. In the group injected with 10 5 cells one animal from G1 died 1 day
before the examination.
In the group injected with 106 cells, three animals died, two animals before the examination
from CG and G1 respectively and one animal two days before the examination from G2.
47
Figure 10: effect of pneumoperitoneum upon tumorimplantation and with addition of 3% oxygen
Experiment D. Effect of 60 min of pneumoperitoneum upon RENCA cells implantation.
In comparison with control group, tumor implantation increased after one hour of pure CO2
pneumoperitoneum for all doses used. This increase was found for total number of tumor
nodules (Figure 10) and for number of nodules in mesentery and abdominal wall (Table 2)
(3x104: Mesentery: P= .03, Abdominal wall: P= .02, Total: P= .03; 105 evaluated after 14 days:
Mesentery: P= .03, Abdominal wall: P= .03, Total: P= .03 and 105 evaluated after 21 days:
Mesentery: P= .03, Abdominal wall: P= .03, Total: P= .03). In comparison with pure CO2
pneumoperitoneum, tumor implantation decreased with the addition of 3% of oxygen to the
CO2 pneumoperitoneum for all doses used. This decrease was found for total number of
tumor nodules and for number of nodules in mesentery and abdominal wall (3x10 4:
Mesentery: P= .03, Abdominal wall: P= .03, Total: P= .03; 105 evaluated after 14 days:
Mesentery: P= .03, Abdominal wall: P= .03, Total: P= .03 and 105 evaluated after 21 days:
Mesentery: P= .03, Abdominal wall: P= .03, Total: P= .03). In comparison with control group,
tumor implantation increased after one hour of CO2 and 3% of oxygen for all doses used.
This increase was found for total number of tumor nodules and for number of nodules in
mesentery and abdominal wall (Table 2) (3x104: Mesentery: P= .03, Abdominal wall: P= .02,
48
Total: P= .03; 105 evaluated after 14 days: Mesentery: P= .03, Abdominal wall: P= NS, Total:
P= .03 and 105 evaluated after 21 days: Mesentery: P= NS, Abdominal wall: P= .03, Total: P=
NS).
IV. Nitrous oxide
Mailova K, Corona R, Verguts J, Adamian L,Koninckx P. The addition of N2O to the CO2
pneumoperitoneum reduces adhesion formation in a laparoscopic mouse model. Manuscript
prepared
AIM:
To
investigate
the
effect
of
different
concentrations
of
nitrous
oxide
pneumoperitoneum on adhesion formation in a laparoscopic mouse model.
MATERIALS & METHODS: As was stated before (cfr. supra) N2O may have a beneficial effect
on the mesothelial barrier and thus on adhesion formation. The exact concentration to
obtain these beneficial effects was never established.
All mice in this experiment received 4 standard bipolar lesions and one hour of
pneumoperitoneum as described above. Pneumoperitoneum with variable concentrations of
N2O was established by two insufflators (Thermoflator Plus, Karl Storz, Tüttlingen, Germany):
one for insufflation of CO2 and one for insufflation of N2O. To obtain a homogenous mixture,
the output of both insufflators was mixed in a mixing chamber which was connected to a
water valve with free escape of gas for a continuous flow and insufflation pressure. The
insufflation pressure for both insufflators was set at 16 mm Hg. Mixture with 5% of nitrous
oxide in carbon dioxide was achieved by 9,5 l/min of CO2 and 0,5 l/min N2O, similarly 10% of
N2O by 4,5 l/min CO2 and 0,5 l/min N2O, 25% of N2O by 3,0 l/min CO2 and 1,0 l/min N2O, 50%
of N2O by 1,0 l/min CO2 and 1,0 l/min N2O.
Experiment I (n=18) was a designed as an exploratory pilot experiment evaluating 100% CO2
(group I; n=9) to 100% N2O (group II; n=9) upon adhesion formation. Two animals died in
group II during the procedure because of intubation problems.
Experiment II (n=30) was as a dose finding study, to establish the lowest effective
concentration of N2O upon prevention of adhesion formation. Six groups (n = 5 in each
group) were used with 100, 50, 25, 10, 5 and 0% N2O.
49
Statistical analyses were performed with the SAS software (SAS System, SAS Institute, Cary,
NC) using the non-parametric Kruskal-Wallis.
RESULTS: Experiment I demonstrated that 100% N2O for the pneumoperitoneum instead of
CO2 decreased adhesion formation, both the quantitative score (p<0,012), and the
qualitative score. Total adhesion score, type, tenacity and extent decreased from 3,0±0,3 to
1,6±0,4 (p<0,009), from 10,8 to 5,5 (p<0,023), from 10,8 to 5,6 (p<0,025) and from 11,3 to
4,9 (p<0,005) respectively.
Experiment II confirmed the effect of 100% N2O on adhesions while surprisingly the effect
persisted until as little as 5% of N2O was used. Indeed the proportion of adhesion formation
decreased (Figure 11) already with addition of 5% of N2O to the CO2 pneumoperitoneum
(P=0,0138). The same decrease was observed for the total adhesion scores being 2,6 ±1,4;
0,7 ±0,9; 1,0±0,3; 1,8±0,8; 0,5±0,7; 0,4±0,9 for groups VI to I respectively. Significances in
comparison with pure CO2 were P=0,0339, P=0,0259, NS, P=0,0150 and P=0,0236. Proportion
scores were significantly lower in groups I, II, IV and V compared to group VI where pure CO2
was used for insufflation, being significant (P=0,0634 ), (P=0,0138 ), (P=0,0245), (P=0,0138)
respectively.
Figure 11: Effect of the addition of different proportions of nitrous oxide to CO2 pneumoperitoneum
on adhesion formation, scored 7 days after surgery. Significances in comparison with pure CO 2 are
indicated: * P<0.05, ** P=0.01
50
V. Depth of Hypoxia
Verguts J,Binda M, Corona R, Koninckx P. Manuscript in preparation
Aim: to investigate the depth of hypoxia caused by the pneumoperitoneum with pure CO2
and after addition of 3% of oxygen to the pneumoperitoneum
MATERIALS & METHODS: The depth of the hypoxia caused by the CO2 pneumoperitoneum
was measured by the Hypoxyprobe™-1 immunoperoxidase staining for hypoxia.
Hypoxyprobe™ kits consist of two parts. The first part is a substituted 2-nitroimidazole
whose chemical name and only ingredient is 1-[(2-hydroxy-3-piperdinyl)propyl]-2nitroimidazole hydrochloride which was injected in the peritoneum before surgery. The
second part contains a primary antibody reagent that recognizes Hypoxyprobe adducts in
hypoxic cells for which it is stained after fixation. Biopsies from the peritoneum were
formalin-fixed, paraffin-embedded and a peroxidase immunohistochemistry technique was
used for staining.
Three situations were examined: control without laparoscopy, laparoscopy with 60 minutes
of carbon dioxide pneumoperitoneum and laparoscopy with 60 minutes of carbon dioxide
with 3% oxygen. Samples were taken from the peritoneum and stained with Hypoxyprobe™1 immunoperoxidase. Slides were evaluated for hypoxia in the mesothelial and
submesothelial layer. Hypoxic cells should stain brown for hypoxia. (Picture 2)
RESULTS: The mesothelial layer clearly stained for hypoxia after 60 minutes of
pneumoperitoneum with CO2. (Picture 2, arrow) There was no staining in the control without
laparoscopy or when 3% of oxygen was added to the CO2 pneumoperitoneum. (Picture 2)
These data confirm that pure CO2 creates mesothelial hypoxia as concluded from previous
experiments on adhesion formation. These experiments furthermore, demonstrate that the
depth of hypoxia is limited and confined to the mesothelial layer.
51
A
B
C
D
Picture 2: A. Hypoxia control example from Hypoxyprobe™-1: hypoxic cells are colored brown
(peroxidase staining), B. Control without laparoscopy, C. Staining after 60 minutes of CO2
pneumoperitoneum, D. Staining after 60 minutes of CO2 pneumoperitoneum + 3% oxygen
52
B. Clinical studies
I. Adding 4% oxygen to the pneumoperitoneum
a. CO2 absorption
Verguts J, Corona R, Vanacker B, Binda M, Koninckx P. Adding 4% of oxygen to the CO2
pneumoperitoneum decreases CO2 absorption. Manuscript prepared
AIM: Addition of 4% of oxygen to the pneumoperitoneum will prevent mesothelial hypoxia
and will decrease CO2 absorption.
MATERIALS & METHODS: In this prospective randomized trial using pure CO2 or CO2 + 4% of
oxygen we included twenty women undergoing laparoscopy for at least one hour, N=10 for
each subgroup. Laparoscopy was standardized by adjusting the insufflation pressure exactly
to 15 mm of Hg. A Trendelenburg position of 30° was maintained. If during surgery more
Trendelenburg or a higher insufflation pressure would be required, the patient would not be
used in the analysis.
Endpoints of the trial were heart rate, blood pressure, ventilation frequency, stroke volume
and end tidal CO2 and they were recorded every minute for the duration of the experiment.
Arterial PaCO2 was measured at the start and after 60 minutes of surgery. The area of
surgical deperitonealization was clinically estimated every 10 minutes.
Following induction of anesthesia, a pneumoperitoneum was created and parameters (i.e.
end tidal CO2, blood pressure or heart rate) were recorded in 20 women during laparoscopy
for at least 60 minutes.
RESULTS: Regarding systolic blood pressure and heart rate there were no significant
differences between both groups. The initial raise in systolic blood pressure was
approximately 30% and was comparable for both groups. (Figure 12) Also heart rate did not
alter between the two groups.
53
Systolic BP
150
125
mmHg
100
75
50
Diastolic BP
25
0
-25
0
25
50
75
100
125
150
Minutes
Figure 12: Changes in systolic and diastolic blood pressure during laparoscopy with CO2
pneumoperitoneum (black) or with PP + 4% oxygen (red) after creation of the peritoneum (time=0)
Arterial oxygenation at the beginning of surgery or after 60 minutes of pneumoperitoneum
did not show a difference between the two groups, indicating an optimal anaesthesiologic
correction during laparoscopy. (Data not shown)
End tidal CO2 (ET CO2) had a comparable increase in the two groups of about 20% during the
first 15 minutes of laparoscopy. (Figure 13) In the control group where 100% CO2 was used, a
further increase in ET CO2 was seen. In the group where 4% of oxygen was added to the CO2
pneumoperitoneum we noted a significant (p<0.001) decrease in ET CO2 during the rest of
the procedure. The two groups showed a remarkable divergence in ET CO2 starting only 15
minutes after creation of the pneumoperitoneum.
54
Figure 13: End tidal CO2 during laparoscopy with CO2 pneumoperitoneum (black) or with PP + 4%
oxygen (red) after creation of the peritoneum (time=0)
Minute volume had a comparable increase of about 25% during the first 15 minutes of
laparoscopy. (Figure 14) After 15 minutes of laparoscopy we also noted a significant
divergence of the minute volume in favor of the group where 4% of oxygen was added to the
CO2 pneumoperitoneum (p<0.0001). After 60’ the groups were no longer comparable as the
number of women still having a pneumoperitoneum was low.
Figure 14: Minute volume measured during laparoscopy with CO2 pneumoperitoneum (black) or with
PP + 4% oxygen (red) after creation of the peritoneum (time=0)
55
Conclusion: The addition of 4% of oxygen to the CO2 pneumoperitoneum decreases the
progressive increase in CO2 resorption during laparoscopic surgery.
b. Pain and inflammation
Verguts J, Corona R, Vanacker B, Binda M, Deprest J, Koninckx P. Adding 4% of oxygen to the
CO2 pneumoperitoneum decreases pain and inflammation in the post-operative phase after
laparoscopic sacrocolpopexia. Manuscript prepared
AIM: Addition of 4% of oxygen to the pneumoperitoneum will prevent mesothelial hypoxia
and decrease post-operative inflammation and pain.
MATERIALS & METHODS: All patients undergoing a laparoscopic colpopexia from May 2008
till November 2009 at least 18 years of age were eligible for this trial. In order not to have
any interference with other inflammatory conditions or pre-existing pain, women would be
excluded as stated above or in case of chronic pain (i.e. peripheral neuropathy, pathology of
the vertebral column, osteo-articular disease) or acute pain (i.e. trauma).
This was a randomized, double-blind, controlled study. Randomization 1:1 into one of the
two groups (CO2 or CO2 + 4% oxygen) was based on block randomization using a sealed
envelope system of 6 envelopes per block. The envelopes were kept sealed until induction of
anesthesia. The envelope was opened and the appropriate gas was used for the laparoscopy.
The details of the study were kept blinded to the investigator that collected the postoperative data. Patients were discharged as soon as they were adequately ingesting orally
and were mobile.
All operations were carried out by the same team of surgeons. Laparoscopy was
standardized by adjusting the insufflation pressure exactly to 15 mm of Hg in all patients. A
Trendelenburg position of 30° was maintained. If during surgery more Trendelenburg or a
higher insufflation pressure would be required, the patient would not be used in further
analysis.
Promontofixation started by dissecting the peritoneum of the promontory, paracolic gutter
up to the vaginal vault and the posterior vaginal vault up to the level of the levator ani
56
muscle. A polypropylene mesh was sutured anterior and posterior to the vault by PDS 0
sutures (Ethicon, Inc.) and fixed at the level of the promontory by stapling. The peritoneum
was closed using a monocryl 3/0 (Ethicon, Inc.) running suture.
Anesthetic technique: An intravenous (IV) cannula was inserted into a vein of the forearm
for the administration of anesthesiologic drugs and fluids. Standard monitoring consisted of
electrocardiogram, non-invasive blood pressure measurements, and pulse oximetry, as well
as end tidal CO2 and sevoflurane (%) measurements.
Anesthesia was induced with an IV opioid (choice was left to the discretion of the
investigator) and IV propofol, and maintained using sevoflurane at 1–2 minimum alveolar
concentration (MAC) end tidal and opioids, according to the patient’s need. After induction
of anesthesia, a neuromuscular blocking agent was administered to facilitate orotracheal
intubation. During 2-3 minutes patients were ventilated through a face mask with oxygen/air
at normocapnia and than intubated; a nasogastric tube was inserted. Neuromuscular block
was monitored using a TOF-watch (Organon-Oss, The Netherlands). Prophylactic postoperative nausea and vomiting medication was provided.
Central body temperature was maintained at ≥35ºC (Bair Hugger). Heart rate and
noninvasive blood pressure measurements were performed continuously and recorded.
Post-anesthetic heart rate, arterial blood pressure, oxygen saturation and respiratory rate
were monitored as part of clinical routine in the recovery room.
Peri-operative analgesia: The local analgesic protocol as designed by the department of
anesthesiology (J. De Coster MD and E. Vandermeulen MD, 2006) was used for all patients.
1/ all patients will be loaded in the operating room with
Perfusalgan® (max. 2000mg):
30
mg/kg IV
Taradyl®:
0.5
mg/kg IV
Dipidolor®:
0.05-0.1
mg/kg IV
Perfusalgan® (max. 1000mg):
15
mg/kg IV / 6 h.
Taradyl®:
0.25
mg/kg IV / 6h
>6y:
0.2-0.3
mg/kg IM
>80y:
0.15-0.2
mg/kg IM
2/ in the PAZA therapy is continued
Dipidolor®:
Limitation in the use of Perfusalgan®, Taradyl® or Dipidolor® as described by the manufacturer.
57
For each patient, age, body mass index (BMI) and duration of surgery (from incision to
closure of the laparoscopic ports) were recorded. To measure pain intensity, the Visual
Analogue Score (0 – 100 mm scale) pain score was measured by a blinded investigator at 4,
24, 48 and 72 hours after surgery to asses pain intensity in the shoulder, trocar wound,
subcostal and visceral pain. VAS pain scores of 30 mm or less are categorized as mild, scores
from 31-69 mm as moderate and scores of 70 mm or more as severe pain.
A blood sample was taken the day before surgery and on day 1, 2, 3 and 4 after surgery as is
standard in our centre for patients undergoing a laparoscopic colpopexia. C-reactive protein
(CRP), white blood cell count (WBC) always expressed as 109/L and CA-125 always expressed
as kU/L was evaluated.
Statistics: Statistical analysis was performed with the SAS System (SAS institute, Cary, NC)
using the nonparametric Kruskal-Wallis test to compare individual groups. All data are
presented as the mean ± SE.
RESULTS: Patients (n=24) were randomized to pneumoperitoneum with carbon dioxide or to
pneumoperitoneum with carbon dioxide + 4% oxygen.
Groups were comparable for age, BMI, laparoscopic operating time and pre-operative scores
for pain, CRP, WBC’s and CA 125. (Table 3)
Table 3: comparison of women having laparoscopy with CO2 pneumoperitoneum (control group) or
with PP + 4% oxygen (oxygen group)
58
Pain-scores in the groups where oxygen was added to the CO2 pneumoperitoneum was
significantly lower (p<0.03) compared to the control group on the day of surgery (day 0).
(Figure 15) The first day after surgery the difference was not statistically significant anymore.
VAS scores after laparoscopic sacrocolpopexia
VAS scores
35
30
control
25
4% oxygen
20
15
10
5
0
day -1 day 0 day 1 day 2 day 3 day 4
days after surgery
Figure 15: General VAS scores for group 1 (oxygen) and group 2 (carbon dioxide): mean and SEM.
Inflammatory reaction as defined by CRP and WBC showed no significant decrease in the
group where oxygen was added to the CO2 pneumoperitoneum. Levels of CA-125 were not
different between the two groups. The means for CRP were however twice as high in the
control group. (Figure 16)
CRP after laparoscopic sacrocolpopexia
60
CRP (mg/L)
50
control
4% oxygen
40
30
20
10
0
pre-op day 1
day 2
day 3
day 4
days after surgery
Figure 16: Means and SEM for C-reactive proteine (CRP) before surgery (pre-op) and the first 4 days
after.
59
The use of pain killers after surgery was not significantly different between the two groups
although less pain medication was used by the study group receiving oxygen on all postoperative days. The difference was the greatest for the use of paracetamol at the day of the
surgery (p<0.09) and at the second day after surgery for ketorolac (p<0.16).
Conslusion: The addition of 4% of oxygen decreases postoperative pain and CRP at least on
day 1-2 after surgery.
c. Port-site metastases
Verguts J, Vergote I, Amant F, Moerman P, Koninckx P. The addition of 4% oxygen to the
CO(2) pneumoperitoneum does not decrease dramatically port site metastases. J Minim
Invasive Gynecol. 2008 Nov-Dec;15(6):700-3. - addendum
AIM: Addition of 4% of oxygen to the pneumoperitoneum will prevent mesothelial hypoxia
and will decrease tumor implantation in laparoscopic port sites.
MATERIALS & METHODS: For detection of port-site metastases (PSM) after diagnostic
laparoscopy with or without addition of 4% of oxygen to the carbon dioxide
pneumoperitoneum all patients presenting with an ovarian mass, suspected to be malignant
were included (n=35). Of these 22 fulfilled the criterium of malignancy with the necessity of
a second look laparotomy. 13 women were excluded since two had an endometrial cancer,
two did not have a malignancy whereas in nine no primary or interval debulking was
performed.
An exploratory prospective randomized controlled trial was initiated using pure CO2 or 96%
CO2 + 4% of oxygen, using the Thermoflator plus for insufflation (Karl Storz Tuttlingen).
Pressure was standardized at exactly 15 mm Hg. At subsequent debulking, the laparoscopic
ports were excised and examined by the pathologist for PSM. PSM were defined as the
presence of at least 1 microscopic cluster of tumor cells observed in a single microscopic
slide made from the port site. Body mass index, type of tumor, stage according to the FIGO
classification and duration of laparoscopy were used for subgroup analysis.
60
This trial was not powered to detect minor decreases in port site metastases. Indeed
considering the 17% incidence of PSM, 192 women would be needed to detect a 50%
reduction. Therefore an interim analysis after one year was done in order to evaluate
continuation.
Analysis was done with the SAS system, with the use of a Spearman r correlation.
RESULTS: As expected both groups were comparable for demographic data as length,
weight, BMI, duration of surgery, type of tumor and pathology. No major complications such
as bowel perforations, bowel fistula, major bleedings, or infections were observed.
The interval between diagnostic laparoscopy and initiation of therapy (primairy debulking or
neo-adjuvant chemotherapy) was 18 days for the control group (n=11) and 12 days for the
study group (n=11). In the control group 19 port sites were excised, and 9 (47%) showed
microscopical PSM. In the oxygen group 16 port sites were excised and 8 (50%) had PSM. In
both groups 5 women had PSM (45%). PSM were not higher in the 12 millimetre umbilical
port than in the 5 millimetre ports. PSM were not higher in ports through which specimens
or biopsies had been extracted.
PSM correlated with the tumor grade (P<0.05) and negatively with length (P<0.018), but not
with BMI. Also after correcting for these variables oxygen addition did not have any effect on
the incidence of PSM
Conclusion: The addition of 4% of oxygen to the CO2 pneumoperitoneum does not decrease
port site metastases.
d. Ultramicroscopic alterations in laparoscopy
Riiskjaer M, Binda M, Verguts J, Corona R, Forman A, Koninckx P. Peritoneal morphology
after laparoscopic surgery in a mouse model. Manuscript prepared
AIM: Addition of 4% of oxygen to the pneumoperitoneum will prevent mesothelial hypoxia
and prevent retraction of the mesothelial cells and exposure of the extracellular matrix.
MATERIALS & METHODS: Women undergoing a laparoscopic sacrocolpopexia were eligible
and randomized in this trial.
61
After installation of the pneumoperitoneum a biopsy of approximate 1cm² was taken and
fixated in 2% formaldehyde + 2,5% glutaraldehyde in Sörensen buffer. Further processing
consisted of rinsing in Sörensen buffer without fixative and placement in 1% osmium for 1
hour. Further dilution in Sörensen buffer and dehydration in ethanol 50-70-90-100 % (each
percentage for 3 x 20 minutes) and gradually replacing by acetone. The ethanol/acetone was
removed and pure HMDS was added and removed. The sample was mounted on aluminum
stubs with silver paint, dried thoroughly and coated with platinum. All samples were viewed
by an experienced lab worker.
RESULTS: After 4 patients the trial was stopped early as specimens obtained in this trial were
not representative. No mesothelial cells were visible in the pre- or post-pneumoperitoneum
biopsies and only fibrous tissue was seen at scanning electron microscopy (SEM). (Picture 3)
A
B
Picture 3: A. SEM of human peritoneum after 60 minutes of pneumoperitoneum with CO2 + 4%
oxygen B. SEM of mouse peritoneum after 60 minutes of pneumoperitoneum with CO2 + 3% oxygen
The SEM pictures obtained in animals by the same technique were showing some effect of
the addition of 3% of oxygen to the pneumoperitoneum. Pure CO2 pneumoperitoneum had a
deleterious effect on the mesothelial morphology and the effect increased with the duration
of the pneumoperitoneum. This was observed immediately after the pneumoperitoneum
and after 24 hours. When 3% oxygen was added to the 60 minutes pneumoperitoneum with
humidification of the gas, mesothelial cells slightly retracted and microvilli were still present.
(Picture 3)
62
e. Simultaneous analysis of inflammation and surgery related complications
when 4% of oxygen is added to the CO2 pneumoperitoneum
MATERIALS & METHODS: We reviewed the effect of 4% of oxygen in all 66 patients from the
3 RCT’s as described above, comprising, 20 from the trial of CO2 absorption, 24 from the trial
on pain and inflammation and 22 from the trial on port-site metastases.
RESULTS: As expected patients were comparable for age, BMI and duration of surgery.
(Table 4)
Parameter
Oxygen group
Control
p-value
Total, n=66
33
33
NS
Age (y), mean +/- SD
51+/- 17
51 +/- 19
NS
BMI, kg/m² +/- SD
25 +/- 5.7
25 +/- 3.7
NS
Duration of surgery (minutes), mean +/- SD
163 +/- 89
203 +/- 113
NS
Time to first flatus (hours), mean +/- SD
32 +/- 18
44 +/- 25
NS
Time to first stool (hours), mean +/- SD
66 +/- 32
61 +/- 29
NS
Table 4: comparison of women having laparoscopy with CO2 pneumoperitoneum (control group) or
with PP + 4% oxygen (oxygen group)
A two way analysis of variance, evaluating simultaneously the effect of the addition of 4% of
oxygen, and the trial (thus compensating for eventual inter trial variability) failed to reach
statistical significance. There was a trend however of a decreased time to first flatus after
surgery being 12 hours earlier in the oxygen group. In women where CRP and WBC were
recorded after surgery (n=56), similarly pain killer intake and CRP were slightly less in the
oxygen group. (Table 5)
63
Parameter
Oxygen group
Control
p-value
Total, n=56
25
31
NS
Ibuprofen (any use)
7
12
p<0.125
Mean +/- SD
Mean +/- SD
CRP pre-operative
2.5 +/- 2.1
2.4 +/- 2.3
NS
CRP day 1
21 +/- 15
33.6 +/- 35.2
NS
CRP day 2
35.8 +/- 31
49 +/- 30
NS
WBC pre-operative
6.8 +/- 2.3
7.3 +/- 2.9
NS
WBC day 1
8.3 +/- 2.1
8.9 +/- 3.5
NS
WBC day 2
7.8 +/- 4.1
9.0 +/- 4.9
NS
Table 5: comparison of women having laparoscopy with CO2 pneumoperitoneum (control group) or
with PP + 4% oxygen (oxygen group) for pain killer intake, C-reactive protein (mg/L) and white blood
cell count (109/L)
Interestingly, in the group with carbon dioxide only three complications occured: two bowel
perforations and one wound eventration with none in the oxygen group (p<0.137).
Conclusion and discussion: although with 4% of oxygen added to the CO 2
pneumoperitoneum, women had less pain, we failed to demonstrate in the entire group a
decrease in CRP, and in time to first flatus. There were no serious adverse events recorded in
the group where oxygen was added to the pneumoperitoneum, adding to the safety of this
altered gas.
The observed difference in complications should be interpreted carefully, since based upon
three events only. If confirmed however, this will be of utmost clinical importance. Although
to a large extent speculative today, we suggest that these complications are not strictly
related to the use of carbon dioxide, but the repair of the peritoneum and/or the viability of
the peritoneum having to cover the wound and allowing for subperitoneal repair processes
may be involved. A viable peritoneum able to cover a superficial wound on the bowel or the
peritoneal wall will protect against macrophages which are present in the abdominal cavity
to adhere to the wound. Macrophages will thus not be able to degrade the matrix covering
the lesion, making a restitutio ad integrum more likely. This may seem to be a paradox as
macrophages are also needed to attract mesothelial cell, but processes in covering a
64
denudated area is speculated to be different from covering a necrotic area. This is in
concordance with the fact that CO2 pneumoperitoneum decreases production of IL-1 and
TNFalfa (decreased inflammation), but increases adhesion formation89.
II. Full conditioning of the peritoneal cavity
a. CO2 absorption
Verguts J, Corona R, Vanacker B, Binda M, Koninckx P, Manuscript in preparation
AIM: Full conditioning of the pneumoperitoneum will prevent mesothelial damage and will
decrease CO2 absorption
MATERIALS & METHODS: In this prospective randomized trial using pure CO2 or full
conditioning we included women undergoing a laparoscopy for an indication of total
laparoscopic hysterectomy, laparoscopic colpopexia, laparoscopic adhesiolysis, laparoscopic
myomectomy or laparoscopic resection of deep endometriosis. CO2 absorption was
measured during laparoscopy by end-tidal CO2 as in the previous trial were only 4% of
oxygen was added to the pneumoperitoneum.
Reduction in CO2 absorption: given previous data that adding 4% of oxygen significantly
reduces CO2 absorption in a group of 20 women undergoing laparoscopic surgery for
endometriosis, a prospective series of 20 patients in each arm (i.e. 40 in total) should be
sufficient to reach statistical significance per stratum.
Women were followed for anesthesiologic changes during surgery i.e. end tidal CO2 and
expiratory N2O, and for inflammatory response (CRP and WBC) and pain as measured by VAS
in the post operative phase. (cfr. infra)
RESULTS: Up to date 20 women were recorded. Measurements showed a significant
decrease in end tidal CO2, even more then with addition of 4% oxygen to the CO2
pneumoperitoneum. The increase in ET CO2 was not as high as in the first trial for both
groups and occurred some 10 minutes later. (Figure 17)
65
Nitrous oxide as measured in the expiratory phase of ventilation did not show any sign of
absorption. The 10% of nitrous oxide present in the insufflation gas thus did not enter the
bloodstream as it was not detectable in the expirated gas.
Figure 17: CO2 resorption during laparoscopy with CO2 pneumoperitoneum (black) or with full
conditioing (red) after creation of the peritoneum (time=0)
The safety of full conditioning was demonstrated for these women. No serious adverse
events occurred during or after surgery.
b. Pain and inflammation
Verguts J, Corona R, Vanacker B, Binda M, Koninckx P, Manuscript in preparation
AIM: Full conditioning of the pneumoperitoneum will prevent mesothelial damage and
decrease post-operative inflammation and pain.
MATERIALS & METHODS: We wanted to demonstrate reduced postoperative pain and a
decreased inflammatory reaction following full conditioning. Secondly we anticipated a
shorter time to resumption of transit i.e. time to first flatus and time to first stool.
66
Postoperative pain was assessed by Visual Analog Scales (VAS). Patients were asked to locate
their pain-symptoms (shoulder, subcostal, trocar wound and visceral pain) and score the
severity. VAS was collected 2-4 hours after surgery and every post-operative day until
discharge. Patients were free to use pain medication after surgery but preferentially
ibuprofen was used in order to permit easier comparison of pain killer intake. Postoperative
inflammatory reaction was assessed by inflammatory parameters as CRP, WBC, and
temperature and were recorded as done routinely. CRP and WBC were measured preoperative and every post-operative day until day 3 or until discharge.
Patients were randomized 1:1. Half of them received standard treatment (CO2
pneumoperitoneum), the other half was randomized to full conditioning of the peritoneum.
In trial 2 women undergoing laparoscopy for total or subtotal hysterectomy, myomectomy,
colpopexia or adhesiolysis were included. Women in trial 1 undergoing laparoscopy for deep
endometriosis were also included, but separately analyzed as they also received
dexamethasone 5mg at the induction of pneumoperitoneum and application by the end of
surgery of Hyalobarrier®gel at the surgical wound.
Surgery and postoperative follow-up were performed as standard. Antibiotics were given as
specific for the surgery performed and immediate post-operative analgesia as is standard.
(see local analgesic protocol, cfr. supra)
A prospective series of 20 patients in each arm (i.e. 40 in total) should be sufficient to reach
statistical significance per stratum. Statistical analysis was performed as an intention to treat
analysis in a 2 way ANOVA. This will allow analysis per stratum and per type of gas used. If in
one procedure the effect of full conditioning is not obtained, this will be visible in the
analysis.
RESULTS: VAS was significant lower in the group receiving full conditioning. (Figure 18) This
was as expected regarding the data from the trials where only 4% of oxygen was used.
Shoulder tip pain 2-4 hours after surgery was significantly lower in the group receiving full
conditioning: 1/22 vs. 11/27 (p<0.0001).
67
Figure 18: General VAS scores (mean and SD) for women having laparoscopy with CO2 PP (square,
red) or with full conditioning (dot, green) for Trial 1 (endometriosis) and Trial 2 (rest)
Difference in CRP was not significant, also not after correction for duration of surgery.
Leucocytosis was unaffected by full conditioning of the peritoneal cavity.
There was a trend of decreased pain killer intake (ibuprofen) in the group with full
conditioning. (Figure 19)
Figure 19: Pain killer intake (mean and SD) for women having laparoscopy with CO2 PP (square, red)
or with full conditioning (dot, green) for Trial 1 and Trial 2
68
c. Adhesion prevention
Verguts J, Binda M, Corona R, Koninckx P. Carbondioxide pneumoperitoneum prevents
postoperative adhesion formation in a rat cecal abrasion model, PhD. J Laparoendosc Adv
Surg Tech A. 2011; author reply, in press- addendum
Koninckx P, Verguts J, Binda M, Vanacker B, Craessaerts M, Corona R. Manuscript in
preparation
AIM: Full conditioning of the pneumoperitoneum will prevent mesothelial damage and
decrease adhesion formation.
MATERIALS & METHODS: Women undergoing laparoscopic excision of deep endometriosis
were randomized in this study since this is severe surgery, associated with severe
postoperative adhesions and since for most of these women fertility is important and hence
a second look laparoscopy can be beneficial. This trial is referred to as trial 1.
40 women should be randomized 1:1. 20 women receiving standard treatment with a CO 2
pneumoperitoneum. 20 women being randomized to full conditioning of the peritoneum
plus rinsing with heparinised saline (5000 U per Liter) plus injection of 5mg dexamethasone
at creation of the pneumoperitoneum plus application of an anti adhesion barrier
(Hyalobarrier Gel Endo®) at the site of surgical wound by the end of surgery.
Second look laparoscopy to assess adhesion formation was performed some two weeks after
surgery under general anaesthesia using a 5 mm laparoscope. Early repeat laparoscopy for
atraumatic lysis of pelvic adhesions is used routinely in many centers for infertility patients.
During this early repeat laparoscopy any existing adhesions were lysed and adhesions were
scored by the surgeon.
11 women were randomized 1:1. 6 women received standard treatment with a CO2
pneumoperitoneum, 5 women were randomized to full conditioning of the peritoneum plus
rinsing with heparinised saline.
Scoring of adhesions: A new scoring system for adhesion formation was developed based
upon the lessons learned from previous classification systems as used in the human and in
69
animal models. Adhesions can be scored quantitatively, as the area of the surgical lesions
covered by adhesions90. This however could only be done in animal models with a well
standardized lesion. Adhesions generally are scored qualitatively by scoring separately
extent, tenacity and type at a given localization91. Past experience in clinical trials and during
surgery, however, made us aware of the strong inter-correlation between extent and
tenacity and type, filmy adhesions generally having low tenacity and covering a small area
only, whereas the rather thin, long, dense and vascularized adhesions are too rare to be
picked up in any classification system. In addition, from the statistical evaluation of adhesion
data we learned that scoring too specifically at too many locations generally is
counterproductive56. Finally scoring systems should be sufficiently practical to permit
immediate scoring after surgery by the surgeon. Otherwise subsequent blind review and
scoring of videorecordings will be necessary. Although this may at first glance be
considered the most objective method, reviewing videorecordings balances between
reviewing and scoring short video clips, and reviewing and scoring the complete recording of
an intervention. The former has the drawback that not all adherences can be grasped in
short videoclips, since the extent of a frozen pelvis can only be judged during and after
surgery. Reviewing complete registrations of the entire intervention, requiring advanced
videocompression and accelerated replay only recently has become technically realistic,
while the time required to review still might be prohibitively long to become practically
realistic.
We therefore scored adhesions as type/tenacity together with 7 areas involved. Type
and tenacity were scored as 0 to 3 when adhesions were absent, filmy with easy lysis and
little or no coagulation, mixed filmy and dense, and dense adhesions requiring sharp
dissection and coagulation respectively. The area involved was scored quantitatively as the
diameter of a theoretical circle comprising all adhesions. This area in fact describes the sum
of all denuded areas caused by adhesiolysis. Filmy adhesions thus almost invariably will
cover a small area, ie often long but very thin. The areas scored separately were kept to a
minimum and were based upon clinical importance and surgical difficulty. Were scored
separately 1. vesico-uterine fold, 2. adhesions between abdominal wall and bowels or
omentum (almost invariably a consequence of previous surgery) and 3. adhesions between
the back side of the uterus and the pouch of Douglas or bowels (as a consequence of
previous surgery, PID of deep endometriosis). Areas 3 and 4 comprised adhesions around
70
left and right ovaries and oviducts together and areas 5 and 6 scored adhesions between
bowels and lateral side-walls in the pelvis. Finally 7 grouped all other adhesions outside the
pelvis without further specification. This was added for completeness.
Statistical analysis and validation of the scoring system: To the best of our knowledge all the
available adhesion scoring systems are based upon common sense, surgical difficulty and
anticipated impairment of fertility but have not been validated. It indeed is unclear whether
the scoring of extent, type, tenacity can be simply added or whether some should get more
weight in order to predict or measure risk of occurrence after surgery, or to
predict recurrence after lysis, or to predict fertility, pain or reoperation. For these trials we
calculated the adhesion load at each site by multiplying scoring and area of denudation
(mm²).
With collection of more data it is the aim to model the relative importance of area involved
and type of adhesions in order to obtain a more predictive total score of adhesions at any of
the clinically important areas.
RESULTS: Although numbers are still small, statistically significant differences between the
two groups were observed. In the control group severe adhesions were systematically
found, whereas in the full conditioning group, adhesions were either minimal or not
existent. (Figure 20)
Figure 20: Adhesions scored (type x area) at the initial laparoscopy (beginning and end of surgery)
and at repeat laparoscopy two weeks later.
71
d. Absorption of fluid from the peritoneal cavity
Koninckx P, Verguts J, Timmerman D. Assessment of measurement validity. Fertil Steril. 2006
Jan; 85(1):268; author reply 268. - addendum
Verguts J, Timmerman D, Bourne T, Lewi P, Koninckx P. Accuracy of peritoneal fluid
measurements by transvaginal ultrasonography. Ultrasound Obstet Gynecol. 2010 May;
35(5):589-92. - addendum
Verguts J, Corona R, Timmerman D, Koninckx P. Absorption of Ringer lactate and Adept after
full conditioning of the peritoneal cavity during laparoscopy. Manuscript in preparation
AIM: Full conditioning of the pneumoperitoneum will prevent mesothelial damage,
decreasing speed of absorption of fluid (Ringer lactate or Adept®).
Measurement of volume
MATERIALS & METHODS: A non invasive method to measure accurately the volume of intraabdominal fluid would be clinically useful to assess retention time of anti-adhesion flotation
agents believed to be a key factor in effectiveness.
A previous trial by Muzii et al.92 only included volumes up to 300 ml and suggested a linear
correlation between volume measured by ultrasound and real volume. We think this
correlation is not linear93 and designed a study to 15 patients undergoing minor laparoscopic
surgery (tubal sterilization, diagnostic laparoscopy) were enrolled after informed consent.
Besides age, also weight, length and BMI were recorded, since these could obviously
influence the collection of larger amounts of fluid in the pelvis. After laparoscopy all
peritoneal fluid was aspirated from the pelvis and carbon dioxide was carefully removed.
The patient was then placed in a 30° reverse Trendelenburg position and a catheter was
placed 5 centimeters through the umbilical port in the direction of the pouch of Douglas
through which Ringer lactate was infused using a Wolf 2220 Hystero Pump® in four discrete
steps of 100, 250, 500 and 1000 ml (increments of 100, 150, 250 and 500mL) i.e. a
logarithmic increase. An equilibration time of 2 minutes was used since in initial pilot
experiments little to no changes occurred after this period. The 3 dimensions of the pocket
72
were measured by ultrasound (Esaote Technos with a 6.5-MHz transvaginal probe). Each
measurement was repeated 3 times with one minute interval and the mean of each
measurement was used for the final calculation. At larger volumes, the fluid spread above
the pouch of Douglas anterior and posterior of the uterus. When this occurred the volume of
the uterus was subtracted from the estimated volume. The volume of the uterus was
calculated from its three dimensions considering the uterus as an ellipsoid (length x height x
depth x pi/6) (8, 9). (see Pictures 8 and 9). At the end of the measurements the instilled fluid
was drained with the catheter.
A
B
Picture 4. A. Transvaginal ultrasound image of the pelvic pocket, without involvement of the uterus,
following infusion of 250 mL of Ringer's lactate. Two dimensions—D1 and D2—are indicated. These
were used together with the third dimension to calculate the volume. B. Transvaginal ultrasound
image of the pelvic pocket, extending above the uterus, following infusion of 500 mL of Ringer's
lactate. The volume of the pocket was calculated from its three dimensions (D1 and D2 indicated),
from which the volume of the uterus was subtracted (D3 and D4 indicated).
Means of the volume measurements performed in triplicate were used to calculate the
relationship between the known instilled volumes and the calculated volumes. Several
models were evaluated using JMP Version 7, SAS Institute Inc., Cary (NC), 2005.
RESULTS: Women (n=15) undergoing minor laparoscopic surgery participated in this trial and
at the end of surgery fluid was instilled in discrete steps and measured by ultrasound.
The measurements of the largest pockets performed after 2, 3 and 4 min of equilibration
were not significantly different, confirming that 2 min of equilibration was sufficient for
reliable estimations. Using untransformed instilled volumes and the corresponding
73
ultrasound-calculated volumes the best prediction of the measured peritoneal fluid volume
proved to be a second degree or quadratic regression curve, providing the best trade-off
between goodness of fit and complexity.
The measured ultrasound volume (Y (mL)) was found to be related to the instilled volume (V
(mL)) by means of the quadratic regression equation: Y = −31.9 + 1.3429 V − 0.0005023V2.
Using this equation the volume V can be estimated from a measured ultrasound volume Y: V
= 1336.7 − (1 723 204.9 − (1990.8421 × Y)) 1/2, for values of Y between 0 and 865.6 mL.
(Figure 21) The fitted quadratic curve was significant (P < 0.0001 for the second-degree
coefficient) and accurate (r2 = 0.89, P = 0.0001).
Figure 21. Quadratic regression curve Y = −31.9 + 1.3429 V − 0.0005023 V2 ( ___) of the volumes
measured by ultrasound (Y (mL)) versus the actual instilled volumes (V (mL)) with 95% prediction
intervals (i.e. the mean ±1.96 times the SD of the individual measurements) (----).
When the sets of measurements obtained in each patient were considered separately, the
coefficient of variation was 7.3% (within-patient variation), which is significantly lower than
the overall coefficient of variation (F (12, 51) = 7.0, p<0.001).
74
The volume of intra-abdominal fluid can be quantified reasonably accurately, at least up to 1
L, by measuring the largest pocket of fluid by transvaginal ultrasonography in a patient in the
30◦ anti-Trendelenburg position for at least 2 min. The procedure was standardized with the
women in this position, with direct instillation, in order to collect most fluid present in the
pouch of Douglas, for which we found an equilibration time of only 2 min.
Since the within-patient variability was found to be only 7.3%, this method can be used to
detect changes in peritoneal fluid volume over time. Indeed, a study involving serial
measurements in individual patients, e.g. when estimating resorption of instilled peritoneal
fluid, would need only 10 patients to detect decreases of 15% in volume at the 0.05
significance level with a power of 80%.
Measurement of absorption
MATERIALS & METHODS: Primary endpoints of this were to evaluate whether the clearance
rate of peritoneal fluid is exponential or linear over time. (absorption rate expected for
Adept ~30-60 ml/h) and to evaluate the role of the mesothelial barrier in this clearance rate.
Knowing that diffusion to and from peritoneal fluid decreases with increasing molecular
weight (MW), our hypothesis is that clearance rate will decrease following peritoneal
conditioning. Indeed a decreased mesothelial trauma (either through hypoxia or through
denudation) and retraction will expose less the basal membrane.
The study was conceived as a Latin Cross. (Table 6)
Control with CO2
Ringer-lactate
Adept®
n = 10
n = 10
n=5
n=5
n=5
n=5
n = 10
Full conditioning
n = 10
Table 6: Study design fluid absorption: Latin Cross
Horizontally: Prospective randomised trial using pure CO2 or full conditioning. 20 women
were randomized 1:1. N=10 for each subgroup. Vertically: Prospective randomised trial using
1000 ml of fluid: Ringer-Lactate or Adept® left in the abdomen following laparoscopic
75
hysterectomy. 20 women were randomized 1:1. N=10 for each subgroup. The volume was
estimated by ultrasound at times 0, 24h, 48h and 72h as described later.
All women undergoing a laparoscopic subtotal hysterectomy were eligible for this trial. No
limitation in body mass index or uterine size was made.
Laparoscopy was standardized by adjusting the insufflation pressure exactly to 15 mm of Hg
in all patients. Rinsing of the abdomen was performed with the randomized product (RingerLactate or Adept®). One liter of the fluid was left in place after surgery.
The first measurements were performed immediately after surgery in order to account for
any differences in instilled fluid. We estimated the residual volume of fluid in the abdominal
cavity in a 30° anti-Trendelenburg position after 1 minute in this position.
In a small pilot-study of 3 patients we estimated the time to have fluid accumulated in the
Pouch of Douglas (data not shown). On the first post-operative day after we instilled Adept®
for the prevention of adhesions we obtained the patients consent to have the ultrasound
performed. Three ultrasonografic measurements after 1, 5 and 15 minutes (9 in total per
patient) in a 30° anti-Trendelenburg position or after some walking down the hall showed no
differences in measured volume. After this we decided to estimate the residual volume of
fluid in the abdominal cavity in a 30° anti-Trendelenburg position after 1 minute.
The volume was estimated by ultrasound immediately after surgery (time 0) and after 24, 48
and 72 hours.
RESULTS: Fluid absorption was fastest in the carbon dioxide group when Ringer lactate was
used: no detectable fluid after 48 hours and the slope of the curve was the steepest. This
was as expected. The slowest resorbtion occurred however in the carbon dioxide group
where Adept® was used where we still had a pocket of fluid after 72 hours. Resorbtion in the
full conditioning group when Adept® was used was slower then when Ringer lactate was
used. (Figure 22)
76
Measured volume by ultrasound (ml)
Fluid absorption
750
700
650
CO2 + Adept
conditioning + Adept
600
CO2 + Ringer
conditioning + Ringer
550
500
450
400
350
300
250
200
150
100
50
0
0
24
48
72
Hours after surgery
Figure 22: Fluid absorption as calculated by consecutive ultrasound measurements, mean and SEM
III. Lavage of the peritoneal cavity
De Cicco C, Corona R, Schonman R, Verguts J, Ussia A, Koninckx P. Extensive abdominal
lavage during laparoscopic discoid excision of 1 deep endometriosis decreases bowel
complications rate. Submitted
AIM: Extensive lavage after laparoscopic surgery will decrease acute inflammation
MATERIALS & METHODS: With the concept of acute inflammation of the entire peritoneal
cavity, we decided to investigate the effect of extensive lavage after full thickness bowel
resection upon postoperative CRP, leucocytosis and the incidence of late bowel perforations.
To assess the effect of lavage on residual peritoneal fluid and cells and subsequent
inflammation, we designed a prospective trial where we compared either standard rinsing of
the abdomen to extensive lavage with 8 liters of saline. Lavage was started in the upper
abdomen with the patient in a 30° anti-Trendelenburg position and continued until the liquid
77
around the liver was completely clear. To achieve this, usually some 4 liters of saline were
required. Subsequently the patient was placed horizontally and the lavage continued in the
lower abdomen.
Lavage of the peritoneal cavity after surgery was performed in 20 women with
randomization to standard lavage or extensive lavage with 8 liters of saline.
Women in the lavage group and in the control group were comparable for age, weight and
duration of surgery.
RESULTS: Postoperative CRP was significantly lower in the lavage group from day 1 onwards
to day 7 (P=0.01). Moreover, in the lavage group, CRP declined faster, being less than 13
mg/l on day 5, whereas in the control group CRP still was markedly elevated on day 6 with a
mean of 22 mg/l. (Figure 23) WBC counts did not show any difference.
Figure 23: Mean and standard error of CRP concentrations following full thickness resection of deep
endometriosis of the rectum in case of extensive lavage with extensive lavage (8 L) or standard (no)
lavage. Overall significance: P=0.01 (repeated measurement ANOVA)
After implementation of extensive lavage of the peritoneal cavity, complications following
full thickness resection of bowel endometriosis such as late bowel perforations disappeared.
We prospectively compared all cases thereafter to historic controls. In 65 patients who
received 8 liters of extensive abdominal lavage, no bowel complications occurred (0/65). In
78
the historical series of 84 patients who received the standard abdominal rinsing, 8 bowel
complications occurred (8/84). (P<0.02 Chi-Square test).
With extensive lavage, CRP is significantly decreased and late bowel perforation did no
longer occur. These finding can be placed in the protection of the mesothelial layer, as debris
and other inflammatory cells, i.e. T lymphocytes94, are washed out of the cavity with
subsequent decreased adherence to the surgical wounds. We hypothesize that if fewer of
these inflammatory cells adhere to the surgical wound, mesothelial cells may cover the
wound completely within a few days, resulting in an increased healing.
IV. pH of irrigation fluid and wet tongue-tip pain
As pain after laparoscopy is multifactorial, and as pH of the different gasses may have an
influence on pain experienced during laparoscopic surgery we wanted to investigate how pH
of irrigation fluids (Saline or Hartman) changed, when put under pressure (15 mm Hg) of
carbon dioxide, nitrous oxide or our mixture of 86% CO2 + 4%O2 + 10% N2O. Pain sensation
when carbon dioxide (i.e. soda water) comes in contact with the mucosa of the mouth is
believed to be the result of the lowering of the pH.
MATERIALS & METHODS: We heated the irrigation fluid (Saline or Hartman) up to 32°C and
measured pH at 1 Atm with an electronic pH meter (pHep®, HI98127, Caprock Developments
Inc. Morris Plains, USA) with a deviation of +/- 0.02. Fluid was then put in a sealed cup and
carbon dioxide, nitrous oxide or our mixture of 86% CO2 + 4%O2 + 10% N2O was insufflated
at 15 mm Hg into the fluid with a continuous outflow through a small needle valve above the
level of the fluid. After five minutes the cup was opened and pH was measured.
To correlate this with sensation of pain, the three gasses were blown on the tip of a wet
tongue of 15 volunteers (health care workers) and pain scores were measured using a visual
analogue scale (0-100).
RESULTS: The pH of the two fluids did not alter when nitrous oxide was blown through the
fluid under pressure and remained 7.0 +/- 0.1. When blowing carbon dioxide through the
fluid in saline or Hartman, pH decreased significant to a mean of 4.2 +/- 0.04 and 5.2 +/- 0.04
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respectively. (Figure 24) Insufflation of the mixture of 86% CO2 + 4%O2 + 10% N2O through
saline or Hartman decreased pH to a mean 4.3 +/- 0.04 and 5.3 +/- 0.04 respectively.
Differences between saline and Hartman are due to an increased buffering capacity of
Hartman.
Pain scores for the different gasses were significantly different, with practically no pain when
nitrous oxide was used. (Figure 25) Pain scores given for carbon dioxide were higher
compared to the gas mixture (p<0,0498).
Figure 24: mean pH and standard deviation of irrigation fluid in open air (air), or under 14mmHg
pressure of carbon dioxide, nitrous oxide or the 86% CO2 + 4%O2 + 10% N2O (mixture)
VAS score
Pain scores 'tip of the tongue'
45
40
35
30
25
20
15
10
5
0
Carbon dioxide Mixture
Nitrous oxide
Figure 25: Pain scores (mean +/- SD) as measured by VAS with insufflation of 100% carbon dioxide,
100% nitrous oxide or the 86% CO2 + 4%O2 + 10% N2O (mixture) on the tip of the tongue.
80
Chapter 4
Discussion and conclusions
A. Understanding the peritoneal cavity
I. The mesothelial layer: a fragile border
A pneumoperitoneum using carbon dioxide creates a hypoxic environment for the
mesothelial cells. In our mouse model the hypoxic effect on the mesothelial cells was clearly
demonstrated. This hypoxia is superficial, but the effects can be profound regarding
adhesion formation.
Prevention of hypoxia of the mesothelial cells is essential but this can be achieved in
different ways. Addition of oxygen to the insufflation gas used during endoscopic surgery as
demonstrated to be beneficial in animals and human is logic. As was demonstrated by
Matsuzaki et al, hyperventilation of the mouse with oxygen may influence adhesion
formation by oxygenation directed through the blood.95 As during laparoscopy the bloodflow
may be reduced due to intraperitoneal pressure, hypoxia may not be prevented and addition
of oxygen to the gas seems essential.
The mesothelial layer is a single cell layer which is subject not only to hypoxia, but also to
damage inflicted by i.e. lavage, manipulation, dessication and too low or too high
temperatures. These detrimental effects were well addressed in our laparoscopic mouse
model, but are more difficult to confirm in the human. Direct proof of decreased damage
upon the human peritoneum by altering the insufflation gas up to date does not exist.
Ultramicroscopic alterations in the peritoneum which is of course the ultimate proof that the
gas used is beneficial to its integrity is not feasible in humans as this would require in vivo
fixation with a toxic product. Previous trials by Volz et al used fixation in vivo to obtain
representative specimens of the peritoneum in animals42.
A larger specimen could also be suggested, but this larger deperitonealisation would have
altered our results on CO2 absorption. New studies to directly look at the integrity of the
mesothelium will be designed in the near future. Alternative ways to show the influence on
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the peritoneum were used in this doctoral thesis such as tumor implantation, CO2
absorption, inflammation, pain en fluid absorption. These are indirect way to show the
effect on the peritoneum, but these end points all are proven reliable alternatives in animal
experiments.
The fact that addition of 4% oxygen to the pneumoperitoneum or lavage completely
prevented late bowel perforations may indicate that inflammatory cells (i.e. macrophages)
play an important role in the healing process of the peritoneum. One may argue that this is
due to the surgical technique, but by adding oxygen to the pneumoperitoneum, we
hypothesize that the mesothelial barrier stays intact and thus is less subject to inflammatory
cells and also produces less exudate. By this reduced inflammation the weakend bowelwall
may heal in a more rapid way without having macrophages interfering with this process. A
viable peritoneum able to cover a superficial wound on the bowel or the peritoneal wall will
protect against macrophages which are present in the abdominal cavity to adhere to the
wound. The same hypotesis on healing can be true for extensive lavage where macrophages
are depleted through this extensive lavage. Macrophages will thus not be able to degrade
the matrix covering the lesion, making a restitutio ad integrum more likely.
In our laparoscopic mouse we saw that prevention of inflammation did indeed prevent
adhesion formation96. This study is not included in my doctoral thesis, but it proves that
inflammation by all means plays a central in the healing process of the peritoneum.
Administration of dexamethasone or addition of 4%oxygen to the pneumoperitoneum did
decrease adhesion formation in a linear way.
Peritoneal infections are not covered by any study so far with this new gas. There may be a
hypothetical risk that aerobic bacteria present in the abdominal cavity may benefit from
these new conditions. It was previously studied however for different gasses. There was a
significant increase in bacteremia in a CO2 pneumoperitoneum group compared with the
laparotomy-only and helium groups at 1 and 2 hours after surgery in a bacterial infection
model from Erenoglu97. The risk of bacterial translocation in an E. coli rat peritonitis model
was increased with insufflation using CO2 or helium, and this effect was more significant at
lower pressures (3 mmHg) than at higher pressures (14 mmHg)98. The results suggest that
CO2 insufflation may promote the growth of intra-abdominal anaerobic bacteria. Such
bacterial growth may lead to intra-abdominal abcess formation or cause localized peritonitis
to develop into generalized peritonitis99. Macrophages and by consequence adhesion
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formation are strongly induced by enteric bacteria and their antigens such as
lipopolysaccharides100. Prevention of this cascade by administration of a recombinant
bactericidal agent with endotoxin neutralizing activities reduced adhesions in a rat cecal
abrasion model100.
The use of another insufflation gas other then CO2 thus seems favorable in order to prevent
transperitoneal bacterial translocation and it was suggested that laparoscopy without
pneumoperitoneum may be preferred in patients with peritonitis. This new gas with or
without full conditioning is therefore as safe as a CO2 pneumoperitoneum or even safer, but
studies are needed.
II. Mesothelial cells in prevention of adhesions
Harvesting and growing mesothelial cells for three weeks is so time consuming that large
scale experiments become difficult.
These experiments confirm and extend the observations that cultured mesothelial cells
obtained after trypsinization of the peritoneal cavity can reduce adhesion formation as
described in rabbits and rats with mesothelial cells obtained from the omentum 28-30. The
effect moreover is not limited to pure surgical models such as the abrasion model29;30, but
also in the CO2 pneumoperitoneum enhanced adhesion model. The effect is moreover dose
dependent. Since we know that following a 60 minute CO2 pneumoperitoneum mesothelial
cells retract thus exposing directly the basal membrane14, we may speculate that injected
mesothelial cells may fill the gaps between the retracted mesothelial cells thus attenuating
the adhesion enhancing effect of the CO2 pneumoperitoneum by preventing the deleterious
effect of the peritoneal cavity. Considering that 100.000 cells are needed for a half maximal
effect, the hypothesis that the cells affect the entire peritoneal cavity seems attractive, as
100.000 cells exceed what is necessary to cover the injury which only measures 1cm by
1.6mm. These 100.000 cells indeed cover 10 cm2 since a confluent culture flask of 25cm²
contains 250.000 cells. That 100.000 cells are similar to the number of cells recovered from
the entire peritoneal cavity of 1 mouse further lends support to the hypothesis that these
cells
affect
the
entire
peritoneal
cavity.
Whether
this
observation
on
CO2
pneumoperitoneum enhanced adhesions can be extended to hyperoxia, desiccation, or
manipulation enhanced adhesions is likely but remains to be established. Indeed considering
83
that the driving mechanisms of all these factors are micro denuded areas all over the
peritoneal cavity the effect of cultured mesothelial cells is expected to be similar in all. A
(surgical) trauma is essential to inititate adhesion formation but these adhesions are
enhanced by factors from the peritoneal cavity, the latter being quantitatively the most
important. The importance of factors in the peritoneal cavity was unequivocally proven by
the observation manipulation in the upper abdomen increased adhesions at the site of the
surgical lesion101. This moreover was recently proven to be mediated by acute inflammation
of the entire peritoneal cavity and also explains that de novo adhesions (outside the surgical
lesion) were never observed.
The mechanisms of adhesion reduction by cultured mesothelial cells can only be speculated
upon. Whereas in pure surgical models, incorporation in the lesion as demonstrated in
rats102 seems logic, in the peritoneal cavity enhanced models, incorporation in between
retracted mesothelial cells of the entire peritoneal cavity cannot be excluded. Moreover, it is
unclear whether the origin of the mesothelial cells and the culture conditions could be
important or whether these cells actively secrete substances in the peritoneal fluid. We even
cannot ascertain that the effect is specific for mesothelial cells since Alpay et al.
demonstrated that also fibroblasts display different immunologic characteristics with an
effect on adhesion formation103.
It is unclear why the cells from the first washing did not grow although remaining viable. The
same culture medium was used as for the detached mesothelial cells. The only difference
between cells from the first washing and cells obtained after trypsination is the fact that the
former were kept for a much longer period in PBS, which indeed may damage mesothelial
cells by making them lose their ability to divide (oral communication S. Mutsaers).
The effect of mesothelial cells in reducing adhesion formation might seem less important in
the pilot experiment (400.000 cells caused a 50% reduction in adhesions) than in the doseresponse experiment where some 100.000 cells caused a 50% reduction in adhesions. This
difference may be a consequence of the fact that cultured mesothelial cells probably stayed
longer in PBS in the pilot study since our experience in handling cells was less at that
moment. Also variability in adhesion formation has to be taken into account. Indeed,
although total adhesion scores were comparable in the two control groups of both
experiments (3.1) the proportion of adhesions was lower in the second experiment (13.5%)
than in the first experiment (19%). Although overall very reproducible, this variability in
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proportion of adhesions ranging from 45% to 15% between experiments had been observed
previously86;104. This indeed prompted us to block randomise by day while standardising all
experimental conditions as temperature, pneumoperitoneum conditions and duration,
anaesthesia and experience of the surgeon as much as possible.
The clinical implications of these experiments are far reaching whereas the use of
mesothelial cells for adhesion prevention becomes attractive. Extensive lavage during and
after surgery might remove besides debris and fibrin also mesothelial cells and
macrophages, and the latter might be detrimental. These animal experiments combined
with clinical observation at least suggest that these effects should be evaluated in detail in
specific experiments. Whereas collecting and culturing mesothelial cells for adhesion
prevention in the same patient is not realistic today, this method could become useful if
fibroblast would be useful, or if some cell lines with similar effects could be developed, i.e.
stem cells. In case mesothelial cells could be preserved from the peritoneal lavage during
surgery as is the case with peroperative cell salvage for blood, this could be a reliable
method to prevent adhesions and the detrimental effects it may cause.
III. Nitrous oxide
The beneficial effect of N2O upon post-operative pain, intra-operative ventilation is well
known, but the underlying mechanism of action remains unclear66, as well as the lowest
effective dose to obtain these advantages.
The low effective concentration of only 5% N2O in our laparoscopic mouse model was
somehow surprising, but this allowed us to implement a safe (<30%) concentration of 10%
N2O in clinical trials.
The mechanism of action of N2O, however has to be different from the effect of oxygen,
since the effect of even 100% of N2O is comparable to the effect of 5%, whereas oxygen
concentrations above 10% clearly increased adhesions73. We therefore anticipated that N2O
and low concentrations of oxygen will have additive effects. This additive effect cannot be
seen in our data with full conditioning, as other factors such as humidification and control of
temperature are interfering.
The central effect on pain of N2O is known to be the result of the stimulation of opioid
receptors, but peripheral nociception in the peritoneum is still under investigation
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IV. Training and experimental models
Training is not only important to improve quality of surgery, but is also directly related to
prevention of adhesion formation as shown in our laparoscopic mouse model. A previous
study performed in rabbits showed that post operative adhesion formation, duration of
surgery and complication rate decreases with the surgeon training expressed by the
consecutive number of procedures performed.
When comparing experienced and non-experienced surgeons, we saw that experienced
surgeons achieved a plateau in an early stage (after 10 procedures) whereas nonexperienced surgeons didn’t achieve the level of experienced surgeons even after 80
consecutive procedures. To obtain a general level of experience it thus seems not possible to
achieve this within one training model, but it may consist of a larger exposure to surgery to
improve skills.
Improved skills by experience probably indicates a less traumatic, more precise and gentle
surgical technique gained by this experience and appears to be important since post
operative adhesion formation was already lower in the ten first procedures within the group
of experienced surgeons compared to non-experienced surgeons. This confirms the data of
the effect of manipulation-enhanced adhesions38 .
Other studies on the effect of CO2 or Helium pneumoperitoneum upon post operative
adhesion formation have shown an important effect of the time of exposure to CO2
pneumoperitoneum upon adhesion formation. So less surgical skills correlates with a longer
operation time and a longer exposure to the CO2 pneumoperitoneum with increased
mesothelial hypoxia.
Different strains of mice showed a different proportion in adhesion formation, suggesting
that a genetic predisposition influences the process of adhesion formation. Recently we
noted that the Balb/c mice in identical control groups over our different experiments had a
different mean number of adhesions. Research confirmed that the company by which the
mice were delivered did change over time. Strain differences have been reported for other
processes involving fibrosis and healing responses such as hepatic, lung, and colorectal
fibrosis 105-107, myocardial and ear wound healing108;109, and bone regeneration110.
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B. Translation to clinical practice
I. Addition of oxygen
The translation of the mouse experiments showing beneficial effects on adhesion prevention
by addition of 4% oxygen to the CO2 pneumoperitoneum started five years ago. Since then
the addition of 4% of oxygen was further improved by full conditioning with addition of 10%
nitrous oxide, humidification and temperature control.
To study the direct effect of this altered gas on the integrity of the peritoneum is not
clinically feasible, but indirect ways of proving our concept is. We looked at secondary
effects of this gas on the mesothelial barrier as CO2 absorption, pain, inflammation and portsite metastases and tried to connect this to the preserved integrity of the bassier.
The results on the addition 4% of oxygen are very clear regarding CO2 absorption. Our study
demonstrated a small but beneficial effect on pain and inflammation with adding 4% of
oxygen to the CO2 pneumoperitoneum during laparoscopy. This effect is thought to be
mediated through different mechanisms.
We have demonstrated that by adding 4% of oxygen to the CO2 pneumoperitoneum the
inflammatory response will decrease. We can hypothesis that as the peritoneal lining will
lose its integrity during laparoscopy, the extracellular matrix will come in contact with the
peritoneal cavity and produce different cytokines that are released into the abdomen. These
cytokines will induce an inflammatory cascade, resulting in elevated CRP and white blood
count and post-operative pain. The effect of the oxygen on the inflammatory cascade can
also be direct through mediation of reactive oxygen species.
In the global understanding of adhesion formation in the peritoneal cavity, this study clearly
adds the value of maintaining a certain homeostasis in the oxygen level of the mesothelial
cells. As the use of oxygen has already demonstrated a beneficial effect on adhesions and on
a parallel inflammatory reaction in mice, one can speculate that the effect on adhesions in
humans will be parallel to the decreased inflammatory reaction which was demonstrated by
this study.
The group which received the oxygen-carbon dioxide mixture did have lower VAS scores and
used less pain medication to maintain mean pain scores less than 3 on the VAS scale.
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This is the first study to demonstrate the pain scores by VAS for a laparoscopic colpopexia.
Pain scores are demonstrated to be generally low, but even then a difference was observed.
This study should be extended to other types of laparoscopic surgery, were more postoperative pain and inflammation is to be expected. The difficulty to overcome will be the
standardization of the study populaition. In our study, the population underwent a
standardized procedure, but for more extensive types of surgery (oncologic surgery,
endometriosis surgery,…) this will be impossible. A large number of patients is therefore
required.
II. Full conditioning of the peritoneal cavity
All findings from previous trials where only 4% of oxygen was added to the CO 2
pneumoperitoneum were confirmed.
End tidal CO2 as a measurement for CO2 absorption further decreased to an almost steady
state after some 20 minutes. From this we may confirm that the mesothelial layer maintains
its integrity. This finding also adds up to the safety of the full conditioning where hypercarbia
is almost unthinkable under these conditions.
Pain was improved by full conditioning with decreased VAS especially during the first postoperative days. This also resulted in a decreased use of pain killers (i.e. ibuprofen). Pain was
examined for the different gasses on the tongue, where the mixture used for full condition
provoked the least amount of pain. Differences in pH when the gasses were blown through
Hartman or physiologic serum did not differ that much to explain the difference in pain as
experienced on the tip of the tongue.
Looking at fluid absorption rates in the different groups, we saw the slowest absorption in
the women who received Adept®, and the fastest absorption when Ringer Lactate was used.
There was however an inversed correlation with the use of full conditioning, where Adept
had an increased absorption when full conditioning was used and a decreased absorption of
Ringer lactate when full conditioning was used.
We can hypothesize that the full conditioning keeps mesothelial cells more intact and thus
providing more active transport of Adept® whereas Ringer is retained with an intact
mesothelial layer, explaining the somehow inversed relation. With the use of carbon dioxide
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and the thus damaged mesothelial layer the Ringer lactate is easily absorbed through third
space where on the other hand there is no more active transport for Adept® possible and
thus retained for a longer period.
III. Oncologic implications
Our laparoscopic mouse model was designed to detect differences in adhesion formation
under different circumstances. Over the years we added progressively, a control of blood pH
which is affected by ventilation rate and tidal volume, a strict control of body temperature
which can vary widely with anaesthesia and the environmental temperature, a strict control
of duration of manipulations such as intubation and initiation of laparoscopy, the duration of
pneumoperitoneum and the type of gas used for the pneumoperitoneum, its humidification,
temperature and the flow rate or leakage through the abdomen. Each of these variables
were found to affect adhesion formation39;41;111. This should be kept in mind when
performing studies on tumour implantation in mice and it seems important that in any study
these technical details should be listed in detail.
Two different cell lines were used in our study to avoid erroneous conclusions through
observations which would be cell line dependent. The cell lines used, CT26 and RENCA, are
moreover well characterized and have been used in different cancer models 112-115. The
number of tumour cells used in these experiments varied from 10 3 to 106. Survival in all
experiments was excellent up to two weeks. The survival of the mice is inversely related to
the number of cells used. With low number of tumour cells (10 3) mice survived up to three
weeks. With high number of cells (106) mice would only survive up to 5 days. However, these
studies did not indicate whether this resulted in a controlled and standardized tumour
growth or whether an uncontrolled tumour spreading was observed. We therefore first
established the optimal dose for our experiments, as the laparoscopic setting with addition
of oxygen is new in oncologic literature. We observed a tumour implantation pattern that
was reproducible in the 3 different animals from each concentration group.
The suspension vehicle for the cells injected could be an important issue but some authors
do not provide any information on this solution116;117. Those studies indicating the
suspension solution, have used either Hanks balanced saline solution (HBSS), phosphate
89
buffered saline solution (PBS) or culture medium113;118;119. One study showed a difference in
the number of tumour implants depending on the solution used for suspension as it seems
that injecting the cells in PBS yields less tumour implants than when culture medium is
used120. We also observed this detrimental effect of PBS when collecting mesothelial cells for
culture from mice compared to collection in culture medium. Mesothelial cells collected in
culture medium survived up to three weeks in contrast to cells collected in PBS which would
not grow. (article submitted) In addition, studies using these tumour cell lines have different
injection routes i.e. intra peritoneal, tail vein, portal vein, subcutaneous or intra splenic and
different methods to assess the outcome, i.e. by directing counting tumour nodules, by
weight or by tumour mass. The data from the studies where tumour nodules were directly
counted was comparable to ours117;118.
Adhesions increase with duration and insufflation pressure of pneumoperitoneum and this
increase is prevented by adding small amounts of oxygen41;54;121. Pneumoperitoneum,
especially at higher insufflation pressure will compress the capillary flow in the superficial
peritoneal layer which together with diffusion of pure CO2 from the abdominal cavity will
create a gradient of hypoxia in the superficial layers. Adding 3% of oxygen to the CO2
pneumoperitoneum may result in a partial oxygen pressure (pO2) of 23 mm Hg, which is
similar to normal intracellular pO2. Insufflation with pure CO2 or helium during laparoscopy
has been shown to induce a hypoxic environment, with oxygen tensions as low as 5 mmHg in
the abdominal wall. In addition, a recent study showed a 82% reduction of blood flow in the
abdominal wall (rectus muscle) after the creation of a 10 mmHg air pneumoperitoneum for
45 minutes in rats as measured using the laser Doppler technique and a concurrent
significant increase in tumour development after the injection of adenocarcinoma cells into
the abdominal wall rectus muscle compared to the control group (no insufflation) 122. A
second study, showed similar results with a 71% reduction of blood flow when CO 2
pneumoperitoneum was induced together with an increase in tumour implantation when
compared to the control group (not insufflated group)123. Suggesting that ischemia as a
factor contributing to an increased risk for tumour implantation and progression after
laparoscopic surgery with pneumoperitoneum.
The hypoxic environment created by the CO2 pneumoperitoneum may cause upregulation of
matrix metalloproteinase (MMP) activity124. These enzymes have the capability of degrading
the extracellular matrix (ECM), which enables invasion of cancer cells, tumour progression
90
and ultimately metastasis to occur125. Also, the in vitro laparoscopic hypoxic environment
reduces both cell-cell and cell-ECM adhesion due to a down regulation in cell surface
adhesion molecules, these effects being readily reversible once normal conditions are
returned126. Scanning electron microscopic examinations confirmed that the induction of a
pneumoperitoneum induces diffuse damage of the entire mesothelial cell layer with
exposure of the basal lamina and extensive repair mechanisms42. The exposure of
extracellular matrix proteins, including laminin, fibronectin, and vitronectin, to the tumour
cell surface is a possible mechanism for increased tumour cell adherence.
Another possible mechanism may be the promotion of intraperitoneal tumour cell growth by
increasing interleukin-1 (IL-1) production of peritoneal macrophages, which are involved
extensively in the repair mechanisms127. Hypoxia could modulate directly or indirectly the
production of cytokines and growth factors by peritoneal mesothelial cells, macrophages
and fibroblasts. Macrophages can exert both negative and positive effects on tumour
growth, which is greatly affected by the microenvironment. Insufflation with CO 2 has been
shown to compromise intraperitoneal macrophage activity and decrease immunologic
responses128. Yet another study demonstrated that laparoscopy with CO2 significantly
reduces plasma tumour necrosis factor-α (TNF) production and increases IL-10 levels,
confirming that CO2 pneumoperitoneum exerts an influence on the local peritoneal
environment129.
In conclusion, this study confirms the role of a CO2 pneumoperitoneum in tumour
implantation and extends supporting data pointing at mesothelial hypoxia as an important
mechanism in tumour implantation and adhesion formation. Further investigations
concerning changes at molecular level under hypoxic laparoscopic environment are
required.
When trying to translate these significant effects on tumor implantation to clinical practice,
circumstances become difficult as most of the important factors controlled in the lab such as
type of tumor, number of tumor cells, time to evaluation and immunologic response of the
patient are no longer determined by us.
Port site metastases (PSM) are one of the consequences following laparoscopic oncologic
surgery. The overall incidence of PSM in this trial, ie 47%, is much higher than reported
previously. This might be related to the study design which was prospective in contrast with
previous retrospective studies, with probably a risk of underreporting. As predictive factors,
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we found a correlation with tumor grade, something not observed before. Although this
seems not to be surprising, it should, however, be interpreted with caution, given the risk of
spurious correlations in a small series. Factors as type and stage of tumor, duration of
laparoscopy and primary debulking therapy or neo-adjuvant chemotherapy were not
predictive for PSM. Stratification for randomisation obviously is imposible since these factors
will only be known after laparoscopy.
The addition of 4% of oxygen to the CO2 pneumoperitoneum unfortunately did not affect
dramatically the incidence of PSM. Given the strong beneficial effects of adding 4% of
oxygen observed in mouse models, we wanted to test the hypothesis of a reduced incidence
of PSM through a direct effect upon the tumor cells exposed to the pneumoperitoneum, or
by an indirect effect through effects upon peritoneal fluid such as an increase of angeogenic
factors as was demonstrated for adhesion formation. We fully realize that the trial was not
powered to detect minor differences, but given the absence of any effect and the high
prohibitive number of patients necessary to detect even differences of 50% we did not
consider continuation of the trial indicated. The pathophysiology of PSM is poorly
understood. With this trial we may at least conclude that the CO2 used for the
pneumoperitoneum is not causally related, given the absence of effect of adding 4% of
oxygen. This trial also did not confirm the hypotheis of direct soiling from ports through
which tumor had been extracted. Aerosolisation and gas leaks, known as ‘the chimney
effect’, also could not be supported, since we did not observe a higher incidence of PSM in
thin women (correlation with BMI not significant) in whom the risk of gas leaks is probably
higher. Also the negative correlation with length could be related to gas leaks although
spurious correlations cannot be ruled out given the borderline significance and the small
series. It needs to be emphasized that in this trial insufflation pressure was strictly
standardized, whereas in retrospective studies the insufflation pressure might have been
increased in obese women.
Whether macroscopical port site metastases affect survival remains debated given the
controversial data reporting no effect and decreased survival130. Whether microscopic port
site metastasis affect survival is unknown, and this series obviously is too small to answer
the question. A fortiori, the effect of adding oxygen to the pneumoperitoneum upon patient
survival in advanced stage ovarian cancer cannot be excluded today. In Balbc mice we
observed a significant decrease in number and size of metastasis following injection of 2
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types of syngeneic tumors, an effect that we interpreted as a consequence of the decreased
trauma upon the mesothelial cell lining. Since besides an effect of the peritoneum, a direct
effect upon tumor cells cannot be excluded the addition of oxygen still might affect tumor
cell implantation and growth in the abdominal cavity and patient survival. In this study we
did not look at this aspect as this is very subjective and would require far more inclusions.
In conclusion, this is a negative trial which nevertheless seems useful reporting, given the
strong effect of adding 4% of oxygen to the CO2 pneumoperitoneum upon tumor
implantation observed in an experimental mouse model and upon adhesion formation.
C. Conclusions and future perspectives
The peritoneum is a delicate organ which is influenced by its environment in a direct way.
Homeostasis of the environment is essential to decrease adhesion formation, post-operative
pain and to minimize anesthesiologic problems.
Some new means of adhesion prevention such as injection of cultured mesothelial cells were
proven in our laparoscopic mouse model and the findings on adhesion prevention from our
model were translated to tumor implantation and most important to clinical practice. Today
it is clear that conditioning of the peritoneal cavity improves recovery after surgery, with
decreases post-operative pain and improves restoration of bowel transit, and CO2
absorption. For other aspects such as adhesions and ultimately fertility and chronic pelvic
pain the beneficial effects are anticipated, as they are also expected for open surgery and
tumor metastasis.
Further clinical development of full conditioning of the peritoneal cavity with a new device
incorporating humidification, temperature regulation and our new gas mixture will make
future trials more feasible. Cooperation with industry (i.e. e-Saturnus®, Storz®, and Fisher
and Paykel®) was and still is essential in the development of this new approach to
laparoscopic surgery, but also the collaboration with anesthesiology (Prof. Dr. B. Vanacker),
surgery (Prof. Dr. F. Penninckx and Prof. Dr. M. Miserez) and Leuven Research and
Development is key to successful trials. When all parties work together in this “Surgery
Valley of Europe”, we can dream of inter university –industry collaboration putting Belgium
on the surgical map.
93
Large randomized controlled trials are still needed to confirm these first promising, but
already significant findings, but these first results make it clear that peritoneal cavity
conditioning is here to stay and will become a new standard of care in surgery.
Preserving is better than restoring and with this new way of looking at and entering the
peritoneal cavity, laparoscopic surgery may step into a new era.
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D. Summary - English
Laparoscopic surgery advanced over the last years as it holds the promise of reduced
invasiveness and thus of reduced adhesion formation. Results on reduced adhesion
formation were however not as good as expected and new approaches to prevent the
deleterious complications such as bowel obstruction, infertility and chronic pelvic pain were
investigated.
Over the last years we developed a laparoscopic mouse model on which preventive
measurements concerning gas (temperature, humidification, oxygen) and other means (i.e.
dexamethasone, barriers) were tested and the most promising were selected. These
favorable actions needed to be brought into clinical practice to reduce adhesion formation
and to bring laparoscopic surgery to a new standard.
Tumor implantation reduced with the addition of 3% of oxygen to the CO2
pneumoperitoneum in our laparoscopic mouse model. There was however no clinical
correlation with reduced port site metastases by adding 4% of oxygen to the CO 2
pneumoperitoneum in women undergoing laparoscopy for ovarian malignancies.
Intraperitoneal injection of cultured mesothelial cells did decrease adhesion formation in a
dose-response dependable way. To bring this approach to clinical practice will need the
development of other means to safely remove, culture and inject mesothelial cells in
humans in the peri-operative phase.
The direct beneficial effect on the peritoneum of the addition of oxygen to the CO 2
pneumoperitoneum was demonstrated in mice with scanning electron microscopy, but
these experiments were not reproducible in humans. Indirect ways to measure the influence
of the addition of oxygen to the CO2 pneumoperitoneum were investigated.
Absorption of CO2 was investigated by the study of end tidal CO2 (ET CO2) where we noticed
a significant decrease when 4% oxygen was added to the CO2 pneumoperitoneum. Perhaps
related to this, was the significant decrease in post-operative pain and inflammation in
women who received the oxygenated gas.
From ongoing mice experiments we learned that full conditioning of the peritoneal cavity
with humidification of the gas at 32°C and with addition 4% of oxygen and 10% of nitrous
oxide further reduced adhesion formation.
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Clinical experiments with full conditioning of the peritoneal cavity studying ET CO2, pain,
inflammation and fluid resorption as we carried out with the addition of 4% of oxygen all
showed a beneficial effect, with even some more pronounced results on pain and
inflammation.
To preserve the integrity of the mesothelial layer, injection of cultured mesothelial cells or
altering the insufflation gas did provide evidence, but also extensive lavage of the peritoneal
cavity did improve inflammatory parameters, suggesting a depletion of inflammatory cells
able to degrade the surgical lesion.
The peritoneum is a delicate organ which is influenced by its environment in a direct way.
Homeostasis of the environment is essential to decrease adhesion formation, post-operative
pain and to minimize anesthesiologic problems.
Preserving is better than restoring and with this new way of looking at, and entering the
peritoneal cavity, laparoscopic surgery may step into a new era.
96
E. Summary - Dutch
Laparoscopische heelkunde kent een belangrijke opmars in de geneeskunde met het doel
om ingrepen minder traumatisch te maken en zo het risico op adhesies te verminderen. De
verwachtingen werden evenwel niet ingelost en de nieuwe manieren om complicaties van
adhesies zoals darmobstructie, infertiliteit en chronische pelvische pijn, te voorkomen,
dienen te worden onderzocht.
Over de laatste jaren ontwikkelde we een laparoscopisch muismodel, waarin verschillende
maatregelen met betrekking tot het gas (temperatuur, bevochtiging, toevoegen van
zuurstof) en andere (bvb. dexamethasone, barriers) werden onderzocht. De meest gunstige
maatregelen werden geïdentificeerd, maar translatie naar de klinische praktijk is nodig om
zo laparoscopische heelkunde naar een nieuwe standaard te brengen.
Tumorimplantatie bij muizen wordt op dezelfde manier beïnvloed als adhesievorming met
een verminderen van de implantatie door toevoegen van 3% zuurstof aan het CO2
pneumoperitoneum. Er is hiervoor echter nog geen klinische correlatie te zien op de
vermindering van poortmetastasen bij vrouwen die een laparoscopie ondergaan wegens een
onderliggende ovariële maligniteit.
Intraperitoneale injectie van gekweekte mesotheelcellen verminderde het aantal adhesies
exponentieel met de dosis. Deze benadering van adhesiepreventie naar de kliniek brengen
vergt echter de ontwikkeling van nieuwe technieken om de mesotheelcellen veilig te
verwijderen, te kweken en opnieuw in te brengen in de perioperatieve fase.
Het directe gunstige effect op het peritoneum door toevoegen van zuurstof aan het CO 2
pneumoperitoneum werd aangetoond bij muizen door scanning elektron microscopie, maar
dit was niet reproduceerbaar bij de mens. Indirecte manieren om het gunstige effect van het
toevoegen van zuurstof aan het CO2 pneumoperitoneum te onderzoeken werden gebruikt.
Absorptie van CO2 werd gemeten met behulp van end tidal CO2 (ET CO2), waar we een
significante daling zagen door het toevoegen van 4% zuurstof aan het CO 2
pneumoperitoneum. Misschien hieraan gerelateerd is de eveneens significante daling van de
postoperatieve pijn en inflammatie bij vrouwen die het geoxygeneerde gas kregen.
97
Uit de doorgaande muizenexperimenten leerden we dat full conditioning met bevochtiging
van het gas op 32°C en met toevoegen van 4% zuurstof en 10% lachgas de adhesies verder
reduceerde.
Klinische experimenten met full conditioning van de peritoneale holte met evaluatie van ET
CO2, pijn, inflammatie en vochtresorptie waren alle gunstig in het voordeel van de full
conditioning, met een zelfs nog gunstiger effect op pijn en inflammatie dan met enkel
toevoegen van 4% zuurstof.
Om de integriteit van de mesotheliale barrière te vrijwaren, kan injectie van mesotheelcellen
of het veranderen van het laparoscopiegas gunstig zijn, maar eveneens kan uitgebreid
spoelen van het abdomen de inflammatoire parameters gunstig beïnvloeden, wat suggereert
dat inflammatoire cellen weggespoeld worden die een chirurgische wonden kunnen
degraderen.
Het peritoneum is een delicaat orgaan dat beïnvloed wordt door zijn omgeving op een zeer
directe manier. Homeostase van de omgeving is essentieel om adhesies, postoperatieve pijn
en anesthesiologische problemen te verminderen.
Beschermen is beter dan herstellen en met deze nieuwe manier om naar de buikholte te
kijken en om er in te gaan, kan de laparoscopische chirurgie in een nieuw tijdperk stappen.
98
Chapter 5
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117. Hirschowitz EA, Naama HA, Evoy D, Lieberman MD, Daly J, Crystal RG. Regional
treatment of hepatic micrometastasis by adenovirus vector-mediated delivery of
interleukin-2 and interleukin-12 cDNAs to the hepatic parenchyma. Cancer Gene Ther
1999;6:491-98.
118. Tsivian A, Shtabsky A, Issakov J, Gutman M, Sidi AA, Szold A. The effect of
pneumoperitoneum on dissemination and scar implantation of intra-abdominal
tumor cells. J Urol 2000;164:2096-98.
119. Kudo M, Asao T, Hashimoto S, Kuwano H. Closed continuous hyperthermic peritoneal
perfusion model in mice with peritoneal dissemination of colon 26. Int J
Hyperthermia 2004;20:441-50.
120. Kollmar O, Schilling MK, Menger MD. Experimental liver metastasis: Standards for
local cell implantation to study isolated tumor growth in mice. Clin Exp Metastasis
2004;21:453-60.
121. Molinas CR, Tjwa M, Vanacker B, Binda MM, Elkelani O, Koninckx PR. Role of CO(2)
pneumoperitoneum-induced acidosis in CO(2) pneumoperitoneum-enhanced
adhesion formation in mice. Fertil Steril 2004;81:708-11.
122. Lundberg O, Kristoffersson A. Pneumoperitoneum impairs blood flow and augments
tumor growth in the abdominal wall - An experimental study in rats. Surg Endosc
2004;18:293-96.
123. Lundberg O, Kristoffersson A. Reduction of abdominal wall blood flow by clamping or
carbon dioxide insufflation increases tumor growth in the abdominal wall - An
experimental study in rats. Surg Endosc 2005;19:720-23.
124. Canning MT, Postovit LM, Clarke SH, Graham CH. Oxygen-mediated regulation of
gelatinase and tissue inhibitor of metalloproteinases-1 expression by invasive cells.
Exp Cell Res 2001;267:88-94.
125. Pross M, Lippert H, Mantke R, et al. A proteinase inhibitor decreases tumor growth in
a laparoscopic rat model. Surg Endosc 2001;15:882-85.
126. Paraskeva PA, Ridgway PF, Olsen S, Isacke C, Peck DH, Darzi AW. A surgically induced
hypoxic environment causes changes in the metastatic behaviour of tumours in vitro.
Clin Exp Metastasis 2006;23:149-57.
127. Jacobi CA, Ordemann J, Bohm B, et al. The influence of laparotomy and laparoscopy
on tumor growth in a rat model. Surg Endosc 1997;11:618-21.
128. Watson RWG, Redmond HP, Mccarthy J, Burke PE, Bouchierhayes D. Exposure of the
Peritoneal-Cavity to Air Regulates Early Inflammatory Responses to Surgery in A
Murine Model. Br J Surg 1995;82:1060-65.
108
129. Jacobi CA, Wenger F, Sabat R, Volk T, Ordemann J, Muller JM. The impact of
laparoscopy with carbon dioxide versus helium on immunologic function and tumor
growth in a rat model. Dig Surg 1998;15:110-16.
130. van Dam PA, DeCloedt J, Tjalma WAA, Buytaert P, Becquart D, Vergote IB. Trocar
implantation metastasis after laparoscopy in patients with advanced ovarian cancer:
Can the risk be reduced? AJOG 1999;181:536-41.
109
110
Chapter 6
Bibliography and curriculum vitae of Jasper Verguts
Publications:
In international peer-reviewed journals
1.
Timmerman D, Verguts J, Konstantinovic ML, Moerman P, Van Schoubroeck D, et al.
Ultrasonography with colour Doppler imaging can replace second stage tests in women with
abnormal vaginal bleeding, Ultrasound Obstet Gynecol. 2003 Aug;22(2):166-71.
2.
Gaarenstroom KN, Kenter GG, Trimbos JB, Agous I, Amant F, Peters AA, Vergote I.
Postoperative complications after vulvectomy and inguinofemoral lymphadenectomy using
separate groin incisions, Int J Gynecol Cancer. 2003 Jul-Aug;13(4):522-7. (Cf. Review study:
Five years retrospective study on vulvacarcinomas 1999).
3.
Vergote I, Amant F, Neven P, Berteloot P, Leunen K, Verguts J. Surgery in pelvic relapses of
gynaecological cancer, ECCO, EJC 2005 oct; 3 (2).
4.
Koninckx PR, Verguts J, Timmerman D. Assessment of measurement validity. Fertil Steril.
2006 Jan;85(1):268.
5.
Deprest J, Claerhout F, Roovers JP, Spelzini F, Schreurs A, Verguts J, De Ridder D. Sacropexie:
kan het ook laparoscopisch? Ned tijdschtift Obstetrie & Gynaecologie, 2006 jun;119: 82-84.
6.
Verguts J et al. Prediction of recurrence after treatment for high grade Cervical Intraepithelial Neoplasia : the role of Human Papillomavirus testing and age at conisation, BJOG
2006, Vol. 113, issue 11, p1303.
7.
Arbyn M, Tulunay G, Ozgul N, Yalvac S, Verguts J, Poppe W, Sankaranarayanan R. European
Union support for a Turkish reproductive health project to assess alternative cervical cancer
screening methods in Sanliurfa (rural south-east Turkey). Eur J Cancer Prev. 2006
Dec;15(6):552-553.
8.
Verguts J et al. Posterior intra-vaginal slingplasty with preservation of the uterus: case report
of a modified surgical technique in a young myelo-meningocele patient. Gynecol Obstet
Invest 2007;63:203-204.
9.
Verguts J et al. HPV induced ovarian squamous cell carcinoma: Case report and review of the
literature, Arch Gynecol Obstet. 2007 Feb 28.
10.
Verguts J et al. Pathologic risk factors for predicting residual disease in subsequent
hysterectomy following LEEP conisation. Gynecol Oncol. 2007, Jul;106(1):273
111
11.
De Cicco C, Ret Davalos ML, Van Cleynenbreugel B, Verguts J, Koninckx PR. Iatrogenic ureteral
lesions and repair: A review for gynecologists. J Minim Invasive Gynecol. 2007 JulAug;14(4):428-35
12.
Housmans S, Moerman P, Amant F, Koninckx Ph, Donders G, Verguts J. Vulvar ulcers: a
differential diagnosis between Behçet’s disease and Lipschütz ulcus. European Clinics in
Obstetrics and Gynaecology 2007
13.
Van den Bosch T, Verguts J, Daemen A, Gevaert O, Domali E, Claerhout F, Vandenbroucke V,
De Moor B, Deprest J, Timmerman D. Pain experienced during transvaginal ultrasound, saline
contrast sonohysterography, hysteroscopy and office sampling: a comparative study.
Ultrasound Obstet Gynecol. 2008 Mar;31(3):346-51.
14.
Schockaert S, Poppe W, Arbyn M, Verguts T, Verguts J. Incidence of vaginal intraepithelial
neoplasia after hysterectomy for cervical intraepithelial neoplasia: a retrospective study. Am
J Obstet Gynecol. 2008 May
15.
Vanderbeke I, Boll D, Verguts JK. Pregnancy and partus in a patient with a spinal cord lesion]
Ned Tijdschr Geneeskd. 2008 May 17;152(20):1169-72
16.
Van den Bosch T, Verguts J, Daemen A, Gevaert O, Domali E, Claerhout F, Vandenbroucke V,
De Moor B, Deprest J, Timmerman D. Reply. Ultrasound Obstet Gynecol. 2008 Jun
20;32(1):119
17.
Veldman J, Vanoudenhove B, Verguts J. Chronic fatigue syndrome: a hormonal origin? A rare
case of dysmenorrhea membranacea. Arch Gynecol Obstet. 2008 Sep 12.
18.
Verguts J, Vergote I, Amant F, Moerman P, Koninckx P. The addition of 4% oxygen to the
CO(2) pneumoperitoneum does not decrease dramatically port site metastases. J Minim
Invasive Gynecol. 2008 Nov-Dec;15(6):700-3.
19.
Claerhout F, Roovers JP, Lewi P, Verguts J, De Ridder D, Deprest J. Implementation of
laparoscopic sacrocolpopexy-a single centre's experience. Int Urogynecol J Pelvic Floor
Dysfunct. 2009 May 29.
20.
van de Vijver A, Poppe W, Verguts J, Arbyn M. Pregnancy outcome after cervical conisation: a
retrospective cohort study in the Leuven University Hospital. BJOG. 2009 Nov 26
21.
Claerhout F, Moons P, Ghesquiere S, Verguts J, De Ridder D, Deprest J. Validity, reliability and
responsiveness of a Dutch version of the prolapse quality-of-life (P-QoL) questionnaire. Int
Urogynecol J Pelvic Floor Dysfunct. 2010 May;21(5):569-78.
22.
De Ridder D, Berkers J, Deprest J, Verguts J, Ost D, Hamid D, Van der Aa F. Single incision
mini-sling versus a transobutaror sling: a comparative study on MiniArc and Monarc slings.
Int Urogynecol J Pelvic Floor Dysfunct. 2010 Mar
112
23.
Verguts J, Timmerman D, Bourne T, Lewi P, Koninckx P. The accuracy of peritoneal fluid
measurements by transvaginal ultrasound. Ultrasound Obstet Gynecol. 2010 Mar 12.
24.
Deprest J, Klosterhalfen B, Schreurs A, Verguts J, De Ridder D, Claerhout F. Clinicopathological
study of patients requiring reintervention after sacrocolpopexy with xenogenic acellular
collagen grafts. J Urol. 2010 Jun;183(6):2249-55.
25.
Koninckx PR, Corona R, de Cicco C, Verguts J, Ussia A. Pudendal neuralgia. J Minim Invasive
Gynecol. 2010 Sep-Oct;17(5):666
26.
Binda MM, Corona R, Verguts J, Koninckx PR. Peritoneal Infusion with Cold Saline Decreased
Postoperative Intra-abdominal Adhesion Formation: Letter to the Editor. World J Surg. 2010
Aug 31
27.
Corona R, Verguts J, Schonman R, Binda MM, Mailova K, Koninckx PR. Postoperative
inflammation in the abdominal cavity increases adhesion formation in a laparoscopic mouse
model Fertil Steril. 2011 Mar 15;95(4):1224-8. Epub 2011 Feb 4.
28.
Verguts J, Coosemans A, Corona R, Praet M, Mailova K, Koninckx P. Intraperitoneal injection
of cultured mesothelial cells decrease CO2 pneumoperitoneum-enhanced adhesions in a
laparoscopic mouse model. Gyn Surg. 2011 Mar
Papers in national scientific journals
1.
Verguts J, Nolens JP, Orye G, Cartuyvels R. Cervixcarcinoom: infectieziekte? (Cervical
carcinoma: infectious disease?) Belg Tijdschr Geneesk. 2002, 58: 1013-1021.
2.
Verguts J, Cryns P, De Sutter Ph, Ide P, Orye G, et al, Premalignant cervical lesions : from
screening to follow-up, Gunaikeia, 2004.
3.
Poppe W, De Sutter Ph, Poppe W, Tjalma W, Verguts J, Weyers S, et al. Cervical screening,
Belg Tijdschr Geneesk 2004.
4.
Tjalma W, De Sutter Ph, Poppe W, Verguts J, Weyers S, et al. Treatment of abnormal cervical
cytology, Belg Tijdschr Geneesk 2004.
5.
De Sutter Ph, Poppe W, Tjalma W, Verguts J, Weyers S, Colposcopy and treatment of
premalignant cervical lesion. Belg Tijdschr Geneesk 2004.
6.
Weyers S, De Sutter Ph, Poppe W, Tjalma W, Verguts J, Follw-up after colposcopy for
abnormal cervical cytology. Belg Tijdschr Geneesk 2004.
7.
Watty K, De Sutter Ph, Poppe W, Tjalma W, Verguts J, Psychological impact of abnormal
cervical cytology and colposcopy on women, Belg Tijdschr Geneesk 2004.
8.
Dierckx I, Verguts J, Timmerman D, Samenvatting lentevergadering, Gunaikeia, 2005.
9.
Verguts J, Poppe W. Het belang van accurate informatie voor het publiek betreffende het
humane-papilloma-virus-vaccin. Editoriaal, Gunaïkeia 2006 mei 11(4): 93.
113
10.
Verguts J, Koninckx P, Penninckx F, Peritoneale adhesies: stand van zaken, Gunaikeia, 2007
11.
Verguts J et al. Pelvic infections of the women: an overview. Belg Tijdschr Geneesk 2007
12.
S. Lambrechts, J. Verguts, W. Poppe Virale seksueel overdraagbare aandoeningen van de
vrouwelijke genitale tractus. Belg Tijdschr Geneesk, 64, nr. 14-15: 764-770, 2008.
13.
K. Van Mensel, W. Poppe, J. Verguts. Pelvische infecties. Een stand van zaken. Belg Tijdschr
Geneesk, 64, nr. 14-15: 759-763, 2008.
14.
Cornelis N, Paridaens R, Neven P, Verguts J, Wildiers H. Recovery of ovarian function after
chemotherapy-induced amenorrhea in a patient receiving an aromatase inhibitor leads to
tumor stimulation. Belgian Journal of Med Onco vol2 issue 2, 107-110, 2008
114
Curriculum vitae
Personal data
Name:
Verguts
Christian names:
Jasper Karel
Date of birth:
March 12, 1974
Place of birth:
Leuven, Belgium
Citizenship
Belgian
Married to:
Eerdekens Sandra
Children:
Lisa (5-9-’01), Anne (26-11-’02), Marit (26-4-’03), Andreas (30-06-‘06)
Education:
1980 - 1992:
Grammar school at the European school in Mol, Belgium
1992 - 1999:
Medicine at the K.U. Leuven., M.D., Graduation 31-7-1999
Courses 1997:
Tropical diseases, Adolescent psychiatry
1998:
Review paper:
‘Verborgen anaplastisch stromaal sarcoom’ with Prof. dr. I. Vergote
1999:
Review study:
Five years retrospective study on vulvacarcinomas (Prof. dr. I.
Vergote en dr. Katja Gaarenstroom)
Specialization Obstetrics and Gynecology:
1.8.1999 - 31.7.2001
Virga Jesse Hospital, Hasselt (B), dr. J.P. Nolens
2001
Interval examination after 2nd year (K.U. Leuven): passed cum magna laude
1.8.2001 – 31.7.2002
U.Z. Gasthuisberg, Leuven, Prof. dr. A. Van Assche
1.8.2002 - 31.7.2003
Catharina Hospital, Eindhoven, dr. P.A. van Dop
1.8.2003 - 31.7.2004
U.Z. Gasthuisberg, Leuven, Prof. dr. A. Van Assche and Prof. dr. I. Vergote
1.8.2004-onward
Gynecologist at UZ Gasthuisberg, Leuven
Sub specialization
uro-gynaecology (2 years) and gynecologic oncology (2 years) till 8/2008
7-2007
Award: Pfizer Educational Grant
7-2008
Doctoral seminar Peritoneal repair and stem cells
Societies and journals:
2000 till present
Active member of the section ‘Premalign cervical lesions’ of the Flemish Group of
Gynecologic Oncology (FGOG)
2005 till present
GSK - CEVEB board-member (Cervarix ® vaccination evaluation board)
1-2006 till present
Member of the Gynecological infectious platform, FRIDOG - FGOG
1-2006 till present
Member of the Gynecological endoscopic platform (GEP), FGOG
5-2006
Initiation of Cervical cancer screening program (VIA / VILI) and treatment in
Cameroon, Njinikom, Catholic Hospital
115
2006 till present
Council member of Gerson Lehrman Group
2008
contribution to “100 Clinical Cases in Obstetrics and Gynecology” by Hodder Arnold
2010 till present
Chairman of the Interest Group in Infectious diseases in ObGyn of the FGOG
Research:
2005 till present
Patricia-trial (co-investigator): HPV vaccine (GSK)
2005 till present
Laboratory work: P. Koninckx: experimental work on adhesion formation,
tumor implantation, mesothelial cells and laparoscopy in mice
2006
PDA / CRF training course + certificate
2006
ERB-trial Wyeth (co-investigator): endometriosis study
2006
Macro version 3.0 training (Organon)
2006 - 2008
NOMAK-trial Organon (co-investigator): oral contraceptive study
2007 - 2009
HORMOS-trial (co-investigator): SERM
2009 - 2010
Rifaximin (co-investigator): Bacterial Vaginosis
2009 - 2010
HPV vaccination trial (Roche) (co-investigator)
2010
Hot Flushes (Merck) (co-investigator)
2010
In-Choice study (Schering-Plough) (principal investigator)
2010
Libido-trial (Bayer-Schering) (co-investigator)
2011 ongoing
LCS12 vs COC, user satisfaction study (Bayer-Schering) (principal investigator)
Teaching / Congress organization:
2001 - ongoing
Faculty and instructor of the yearly colposcopycourse of the FGOG
2004 - ongoing
Faculty and instructor of the biannual vulvacourse of the FGOG
2004 - ongoing
instructor at the Centre for experimental Surgery, CHT (laparoscopic animal models)
2004 - ongoing
training and teaching of assistants and co-assistants
5-2006
Uro-gynaecology and cervical cancer screening, Njinikom, Cameroon
28-30 sept. 2006
Faculty of the 7th PAX meeting, Universiteitshallen, Leuven
2006 - ongoing
6th year medicine: endoscopy in gynecology, urgencies in gynecology
2007
Female Cancer Program – Asia Link (EuropeAid): HPV vaccination
and laparoscopic techniques
28- 31 aug. 2008
Faculty of the 6th European Conference of ESIDOG
4-2009
Faculty of initiation of cervical cancer screening in Kinshasa, Congo
2-2011-ongoing
Faculty and instructor of the yearly infectious diseases in ObGyn of the FGOG
116
Chapter 7
Addenda
117